Targeted gene modification by parvoviral vectors

ABSTRACT

This invention provides methods for obtaining targeted gene modification in vertebrate cells using parvoviral vectors, including adeno-associated virus (AAV). The parvoviral vectors used in the methods of the invention are capable of targeting a specific genetic modification to a preselected target locus in a cellular genome by homologous pairing.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 10/423,604 filed Apr. 24, 2003, which is acontinuation application of U.S. patent application Ser. No. 09/428,172filed Oct. 27, 1999 and now abandoned, and which claims benefit of U.S.Provisional Patent Application No. 60/106,191, filed Oct. 28, 1998. Thisapplication also claims priority to PCT Patent Application No.US98/07964, filed Apr. 20, 1998, which application designates the UnitedStates and claims priority to U.S. Provisional Patent Application No.60/044,789, filed Apr. 24, 1997. Each of these applications isincorporated herein by reference for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant Nos:P01HL53750 and HL03100, awarded by the National Institutes of Health.The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention pertains to the field of targeted modification ofcellular DNA in vertebrate cells by homologous pairing using parvoviralvectors, including vectors based on adeno-associated virus (AAV).

2. Background

Previously known methods for introducing defined mutations intomammalian chromosomes by gene targeting involve transfection,electroporation or microinjection (Smithies et al. (1985) Nature 317:230-234; Thomas et al. (1986) Cell 44: 419-428). These methods, exceptfor microinjection, produce homologous recombination events in only asmall fraction of the total cell population, on the order of 10⁻⁶ in thecase of mouse embryonic stem cells (Doetschman et al. (1987) Nature 330:576-578; Thomas and Capecchi (1987) Cell 51: 503-512). Thus, the routineuse of these methods requires preselection of transformed cells, makingit difficult to apply the techniques to normal cells and in vivoapplications.

Attempts to use transducing viral vectors to overcome these limitationsand achieve chromosomal gene targeting experiments have been performedwith retroviral and adenoviral vectors, but the results were notsignificantly better than can be obtained by transfection, withhomologous recombination occurring in 10⁻⁵ to 10⁻⁶ cells (Ellis andBernstein (1989) Mol. Cell. Biol. 9: 1621-1627; Wang and Taylor (1993)Mol. Cell. Biol. 13: 918-927).

Adeno-associated virus 2 (AAV) is a 4.7 kb single stranded DNA virusthat has been developed as a transducing vector capable of integratinginto mammalian chromosomes (Muzyczka (1992) Curr. Top. Microbiol.Immunol. 158: 97-129). Two thirds of integrated wild-type AAV provirusesare found at a specific human chromosome 19 site, 19q13-qter (Kotin etal. (1991) Genomics 10: 831-834; Kotin et al. (1990) Proc. Nat'l. Acad.Sci. USA 87: 2211-2215; Samulski et al. (1991) EMBO J. 10: 3941-3950).The site-specific integration event is a non-homologous recombinationreaction that appears to be mediated by the viral Rep protein (Giraud etal. (1995) J. Virol. 69: 6917-6924; Linden et al. (1996) Proc. Nat'l.Acad. Sci. USA 93: 7966-7972). While this feature could prove useful insome applications, AAV vectors with deletions in the viral rep gene havenot been found to integrate at this same locus (Russell et al. (1994)Proc. Nat'l. Acad. Sci. USA 91: 8915-8919; Walsh et al. (1992) Proc.Nat'l. Acad. Sci. USA 89: 7257-7261). Southern analysis of integratedrep AAV vector proviruses suggests that integration sites are random(Lebkowski et al. (1988) Mol. Cell. Biol. 8: 3988-3996; McLaughlin etal. (1988) J. Virol. 62: 1963-1973; Russell et al. (1994) supra.; Walshet al. (1992) supra.) and sequencing of integrated vector junctionfragments has confirmed that integration occurs by non-homologousrecombination at a variety of chromosomal sites.

Although the development of integrating vectors based on eukaryoticviruses has made possible the efficient introduction of genes intomammalian chromosomes, there are many situations where it would bepreferable to modify specific chromosomal sequences. Rather than, forexample, introducing a corrected version of gene at a chromosomallocation other than the native locus for the gene, one could correct thedefective allele at the native locus. This ability could eliminateunwanted chromosomal genotypes and avoid position effects on geneexpression. The need for such an ability to modify a preexisting locusis particularly acute in gene therapy, where mutant genes can havedominant effects and tissue-specific controls on expression are oftencritical.

Thus, a need exists for methods of obtaining specific geneticmodification at selected target sites in vertebrate cellular genomes athigh frequencies. The present invention fulfills this and other needs.

SUMMARY OF THE INVENTION

The present invention provides methods of producing a vertebrate cellthat has a modification at a pre-selected target locus. The methodsinvolve contacting the cell with a recombinant parvoviral vector thatincludes a) a targeting construct having a DNA sequence that issubstantially identical to the target locus except for the modificationbeing introduced, and b) all or part of at least one parvoviral ITR or afunctional equivalent thereof. Upon entry of the vector into the cell,homologous pairing occurs between the targeting construct and the targetlocus, resulting in the modifications being introduced into the targetlocus. The modification can include one or more deletions, insertions,substitutions, or a combination thereof. The methods can be used forintroducing a second modification at a second target locus bytransducing a cell with a parvoviral vector that has a second targetingconstruct that is at least substantially identical to the second targetlocus except for the second modification. Additional target loci can bemodified by transduction using parvoviral vectors that have appropriatetargeting constructs. More than one targeting construct can be includedon each parvoviral vector.

Also provided by the invention are vertebrate cells that containspecific genetic modifications at one or more preselected target locithat were introduced into the cells, or ancestors of the cells, bycontacting the cells with a parvoviral vector that has a recombinantviral genome which includes a targeting construct that includes a DNAsequence which is substantially identical to the target locus except forthe modification being introduced. These cells can be cultured in vitro,ex vivo, or can be part of an organism.

The invention also provides methods for introducing a modification of atarget locus in a cell in a vertebrate by contacting a cell ex vivo witha recombinant parvoviral vector that includes a) a targeting constructhaving a DNA sequence that is substantially identical to the targetlocus except for the modification being introduced, and b) all or partof at least one parvoviral ITR or a functional equivalent thereof. Therecombinant parvoviral vector is introduced into the cell, after whichhomologous pairing occurs between the targeting construct and the targetlocus resulting in the modifications being introduced into the cellularDNA at the target locus. The modified cell is then introduced into avertebrate.

In another embodiment, the invention provides methods for making amodification of a target locus in a cell in a vertebrate byadministering to the vertebrate a recombinant parvoviral vector. Theparvoviral vectors used in these methods include a) a targetingconstruct having a DNA sequence that is substantially identical to thetarget locus except for the modification being introduced, and b) all orpart of at least one parvoviral ITR or a functional equivalent thereof.The recombinant viral genome is introduced into the cell, after whichhomologous pairing occurs between the targeting construct and the targetlocus resulting in the modifications being introduced into the cellularDNA at the target locus.

The invention also provides methods of making an animal that includescells that have a modification of a target locus of interest. Themethods involve introducing into a cell from which an animal can bereconstituted a recombinant parvoviral vector that includes: a) atargeting construct which comprises a DNA sequence which issubstantially identical to the target locus except for the modificationbeing introduced; and b) all or a portion of at least one parvoviral ITRor a functional equivalent. Homologous pairing occurs between thetargeting construct and the target locus resulting in the modificationbeing introduced into the target locus. The cell and/or progeny of thecell is then allowed to develop into an embryo and brought to term. Theresulting animals, which can be either transgenic or chimeric animals,are also part of the invention.

In other embodiments, the methods of the invention are used to obtainmodified nuclei that are used in nuclear transplantation. These methodsinvolve using the gene targeting methods of the invention to introduce adesired modification into a target locus in the genome of the cell thatis to serve as the nucleus donor. The nucleus from a cell that has thedesired modification is introduced into a second cell from which ananimal can be reconstituted. This cell is then allowed to develop intoan embryo and brought to term. Again, the resulting animals, which areeither transgenic or chimeric, are also provided by the invention.

The invention also provides methods for introducing recombinationsignals into the genome of cells. These recombination signals, e.g., loxsites, can serve as substrates for recombinases that recognize theparticular polynucleotide sequences (e.g., the Cre polypeptide). Inthese embodiments, the targeting construct includes a recombinationsignal that is flanked by polynucleotide sequences that aresubstantially identical to the target locus. Upon introduction of thetargeting construct into the cell, homologous pairing occurs with thetarget locus, resulting in the recombination signal being introducedinto the target locus.

Also provided by the invention are methods for enhancing the efficiencyof gene targeting. In some embodiments, the recombinant parvoviralvector includes a targeting enhancer. Enhancers can include, forexample, modifications to the polynucleotide such as an adduct, apyrimidine dimer, a deletion of a sugar and/or base or othermodification of the DNA backbone, and the like. Targeting enhancers canalso include polypeptides that are capable of enhancing gene targeting,for example, recombination polypeptides, DNA repair enzymes, and thelike. These can be included within a viral particle, for example.

Other methods provided by the invention for enhancing the efficiency ofgene targeting involve treating a target cell with an agent thatenhances targeting efficiency. These agents include, for example, one ormore of a cell cycle modulator, a DNA repair modulator, a DNArecombination modulator, a modulator of chromatin packaging, aninhibitor of apoptosis, and a DNA methylation inhibitor.

The invention also provides methods for determining the efficacy ofparvoviral vector-mediated gene targeting. These methods involveproviding a cell having an integrated retroviral provirus that includesa defective reporter gene. The reporter gene is defective in that it hasa first mutation that prevents expression of a reporter gene producthaving a detectable phenotype. The retroviral provirus serves as atarget locus that is uniform from cell to cell. A recombinant parvoviralvector is then introduced into the cell. The recombinant parvoviralvector includes at least a polynucleotide subsequence of the reportergene, wherein the polynucleotide subsequence overlaps the location ofthe first mutation but does not include the first mutation. The reportergene subsequence present in the parvoviral vector does not encode afunctional reporter gene product, due either to a second mutation whichprevents expression of a reporter gene product having a detectablephenotype, or because the subsequence does not encode a full-lengthreporter gene product. Homologous pairing occurs between thepolynucleotide subsequence and the reporter gene resulting in correctionof the first mutation and expression of the reporter gene. Theefficiency of gene targeting is then determined by detecting thepresence or absence of the reporter gene product.

Additional embodiments of the invention provide methods to enrich forcells in which gene targeting at a target locus has occurred. Thesemethods involve providing cells having: a) a reporter gene thatcomprises a first mutation which prevents expression of a reporter geneproduct having a detectable phenotype, and b) a target locus at which amodification is desired. Recombinant parvoviral vectors are introducedinto these cells which have the following elements: a) a selectionconstruct which includes a polynucleotide subsequence of the reportergene, wherein the polynucleotide subsequence overlaps the location ofthe first mutation but does not include the first mutation, andcomprises a second mutation which prevents expression of a reporter geneproduct having a detectable phenotype; and b) a targeting construct thatincludes a DNA sequence which is substantially identical to the targetlocus except for the modification being introduced. Next, cells areidentified in which the reporter gene product is expressed. The cellsthat express the reporter gene product comprise a population of cells inwhich homologous pairing has occurred between the polynucleotidesubsequence and the reporter gene resulting in correction of the firstmutation and expression of the reporter gene. This population of cellsis then screened to identify those in which homologous pairing hasoccurred between the targeting construct and the target locus resultingin the modification being introduced into the target locus.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a diagram of the adeno-associated viral vector AAV-SNori,which is described in Example 1. This vector includes two AAV terminalrepeats (TR), a bacterial gene encoding neomycin phosphotransferase(neo) under the control of an SV40 early promoter and a bacterial Tn5promoter, a p15A plasmid replication origin, and a eukaryoticpolyadenylation site. Also shown are the vectors AAV-SNO39 andAAV-SNO648, which contain mutations at by 39 and by 648 of the neo gene,respectively. FIG. 1B is an autoradiogram which shows the results of aSouthern blot analysis of BamHI-digested genomic DNA from G418-resistantHeLa cell clones that had been modified as described in Example 1. Thelane captioned “HeLa” shows hybridization of a neo gene probe to genomicDNA from unmodified HeLa cells, the lane captioned “HSNO39” showshybridization of the probe to genomic DNA from HSNO39 cells that containthree copies of a plasmid that contained the internal portion ofAAV-SNO39, and lanes 1-11 show hybridization of the probe to elevendifferent clones obtained by modifying HSNO39 cells using the parvoviralvector AAV-SNO648.

FIG. 2A is a diagram of the human HPRT locus, as well as the AAV vectorsHPe2/3 and HPe2/3X, which were used to modify the human HPRT locus. Inaddition to the indicated portion of the HPRT gene, these vectors, whichare described in Example 2, contain two AAV terminal repeats (TR), andfour Alu repeats designated 0, P, Q, and R. FIGS. 2B and 2C areautoradiograms of HT-1080 human fibrosarcoma cell genomic DNA that hadbeen digested with HindIII (FIG. 2B) or HindIII plus PvuI (FIG. 2C). Thelanes captioned “HT1080” shows hybridization of the probe shown in FIG.2A to genomic DNA from unmodified HT1080 cells, and lanes 1-13 showhybridization of this probe to genomic DNA from thirteen differentclones that were made 6TG resistant by transduction using the AAV vectorAAV-HPe2/3X.

FIG. 3 shows the results of an experiment, described in Example 2, inwhich AAV vectors AAV-HPe2/3 and AAV-HPe2/3X were used to modify theHPRT locus in HT-1080 cells. The percent of 6TG-resistant cells obtainedis shown.

FIG. 4 presents the results of an analysis of the effect of multiplicityof infection on the frequency with which a defective neo gene present inHeLa cells is corrected by the AAV vector AAV-SNO648. This experiment isdescribed in Example 3.

FIG. 5 shows a comparison of the frequency of neo gene correction inHSNO39 cells obtained using transduction versus transfection asdescribed in Example 4. Transduction was carried out using the AAVvector AAV-SNO648, while transfection was performed using the plasmidpASNO648 (which contains the entire AAV-SNO648 genome), pASNO39 (whichcontains a mutation at base pair 39 of the neo gene) and pASNori2 (whichhas no neo mutation).

FIG. 6 shows the fraction of normal human fibroblasts having a modifiedHPRT gene after transduction using the AAV vector AAV-HPe2/3X asdescribed in Example 5. The fraction of HPRT-modified cells is plottedversus the number of infecting AAV genomes per cell.

FIG. 7 presents the results of experiments in which four differentnormal human fibroblast cultures were transduced with either AAV-HPe2/3(wild-type HPRT gene) or AAV-HPe2/3X (which introduces a frameshiftmutation in the HPRT gene). The percent of HPRT gene modification isshown.

FIG. 8 shows a schematic representation of gene targeting at aretroviral vector target locus.

FIG. 9 shows vectors used for studies of gene targeting by repair of analkaline phosphatase gene. Two retroviral vectors (MLV-LAPSN) are shown.The vector LAP375Δ4 has a 4 by deletion at nucleotide 375 of the APreading frame, while the vector LAP961Δ2 has a two base pair deletion atnucleotide 961. Either of these vectors are introduced into host cellsas a target locus for gene targeting. The AAV vector used for genetargeting (AAV-5′APBss) includes a portion of the AP gene.

FIG. 10 shows a time course of gene targeting using the AP vectorsystem.

FIG. 11 shows a retroviral vector and an AAV vector that are suitablefor use in testing the ability to correct a neo gene by gene targeting.The retroviral vector MLV-LHSNO39, which is introduced into the hostcells as a target locus, has a neomycin resistance gene that contains aninsertion mutation at by 39 of the neo gene. The AAV vector AAV-SNO648,includes a neo gene that has an insertion mutation at by 648 of the neogene.

FIG. 12 shows the results of an experiment in which a mutant AP gene iscorrected by gene targeting in normal (MHF2) and mutant fibroblasts(xeroderma pigmentosum complementation groups A (XPA1) and C(XPCI), andBloom's Syndrome (BS1).

FIG. 13 shows the structure of the mouse β-glucuronidase genomic locus,and AAV vectors that are used in experiments to correct a mutation inthe genomic β-glucuronidase locus.

FIG. 14 shows β-galactosidase transgenes that are introduced intotransgenic animal cells (pCnZSNO, pCnZGSNO (green fluorescent proteinreporter gene), and pCnZAPSNO (alkaline phosphatase reporter gene). AnAAV vector that contains a portion of the β-gal coding sequence is alsoshown.

DETAILED DESCRIPTION Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described. For purposes of the present invention, thefollowing terms are defined below.

The term “cell line,” as used herein, refers to individual cells,harvested cells, and cultures containing the cells, so long as they arederived from cells of the cell line referred to. A cell line is said tobe “continuous,” “immortal,” or “stable” if the line remains viable overa prolonged time, typically at least about six months. To be considereda cell line, as used herein, the cells must remain viable for at least50 passages. A “primary cell,” or “normal cell,” in contrast, refers tocells that do not remain viable over a prolonged time in culture.

The term “cis-active nucleic acid” refers to a nucleic acid subsequencethat encodes or directs the biological activity of a nucleic acidsequence. For instance, cis-active nucleic acid includes nucleic acidsubsequences necessary for modification of a nucleic acid sequence in ahost chromosome, and origins of nucleic acid replication.

The term “constitutive promoter” refers to a promoter that is activeunder most environmental and developmental conditions.

The term “equivalent conditions” refers to the developmental,environmental, growth phase, and other conditions that can affect a celland the expression of particular genes by the cell. For example, whereinducibility of gene expression by a hormone is being examined, twocells are under equivalent conditions when the level of hormone isapproximately the same for each cell. Similarly, where the cell cyclespecificity of expression of a gene is under investigation, two cellsare under equivalent conditions when the cells are at approximately thesame stage of the cell cycle.

The term “exogenous” as used herein refers to a moiety that is added toa cell, either directly or by expression from a gene that is not presentin wild-type cells. Included within this definition of “exogenous” aremoieties that were added to a parent or earlier ancestor of a cell, andare present in the cell of interest as a result of being passed on fromthe parent cell. “Wild-type,” in contrast, refers to cells that do notcontain an exogenous moiety. “Exogenous DNA,” as used herein, includesDNA sequences that have one or more deletions, point mutations, and/orinsertions, or combinations thereof, compared to DNA sequences in thewild-type target cell, as well as to DNA sequences that are not presentin the wild-type cell or viral genome.

The term “homologous pairing,” as used herein, refers to the pairingthat can occur between two nucleic acid sequences or subsequences thatare complementary, or substantially complementary, to each other. Twosequences are substantially complementary to each other when one of thesequences is substantially identical to a nucleic acid that iscomplementary to the second sequence, as defined below.

The term “host cell” or “target cell” refers to a cell to be transducedwith a specified vector. The cell is optionally selected from in vitrocells such as those derived from cell culture, ex vivo cells, such asthose derived from an organism, and in vivo cells, such as those in anorganism.

The term “identical” in the context of two nucleic acid or polypeptidesequences refers to the residues in the two sequences which are the samewhen aligned for maximum correspondence. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith and Waterman (1981) Adv. Appl. Math. 2: 482, by the homologyalignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443,by the search for similarity method of Pearson and Lipman (1988) Proc.Natl. Acad. Sci. USA 85: 2444, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by inspection.

An indication that two nucleic acid sequences are “substantiallyidentical” is that the polypeptide which the first nucleic acid encodesis immunologically cross reactive with the polypeptide encoded by thesecond nucleic acid. Another indication that two nucleic acid sequencesare substantially identical is that the two molecules and/or theircomplementary strands hybridize to each other under stringentconditions.

The phrase “hybridizing specifically to,” refers to the binding,duplexing, or hybridizing of a molecule only to a particular nucleotidesequence under stringent conditions when that sequence is present in acomplex mixture (e.g., total cellular) DNA or RNA. The term “stringentconditions” refers to conditions under which a probe will hybridize toits target subsequence, but to no other sequences. Stringent conditionsare sequence-dependent and will be different in different circumstances.Longer sequences hybridize specifically at higher temperatures.Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (Tm) for the specific sequence at adefined ionic strength and pH. The Tm is the temperature (under definedionic strength, pH, and nucleic acid concentration) at which 50% of theprobes complementary to the target sequence hybridize to the targetsequence at equilibrium. (As the target sequences are generally presentin excess, at Tm, 50% of the probes are occupied at equilibrium).Typically, stringent conditions will be those in which the saltconcentration is less than about 1.0 M sodium ion, typically about 0.01to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 andthe temperature is at least about 30° C. for short probes (e.g., 10 to50 nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Specifichybridization can also occur within a living cell.

An “inducible” promoter is a promoter which is under environmental ordevelopmental regulation.

The term “labeled nucleic acid probe” refers to a nucleic acid probethat is bound, either covalently, through a linker, or through ionic,van der Waals or hydrogen “bonds” to a label such that the presence ofthe probe may be detected by detecting the presence of the label boundto the probe.

The term “label” refers to a moiety that is detectable by spectroscopic,radiological, photochemical, biochemical, immunochemical, or chemicalmeans. For example, useful labels include ³²P, ³⁵S, fluorescent dyes,electron-dense reagents, enzymes (e.g., as commonly used in an ELISA),biotin, dioxigenin, green fluorescent protein (GFP), or haptens andproteins for which antisera or monoclonal antibodies are available.

The term “nucleic acid” refers to a deoxyribonucleotide orribonucleotide polymer in either single- or double-stranded form, andunless otherwise limited, encompasses known analogues of naturalnucleotides that hybridize to nucleic acids in manner similar tonaturally occurring nucleotides. Unless otherwise indicated, aparticular nucleic acid sequence includes the complementary sequencethereof.

The term “operably linked” refers to functional linkage between anucleic acid expression control sequence (such as a promoter, or arrayof transcription factor binding sites) and a second nucleic acidsequence, wherein the expression control sequence directs transcriptionof the nucleic acid corresponding to the second sequence.

The term “recombinant parvoviral vector genome” refers to a vectorgenome derived from a parvovirus that carries non-parvoviral DNA inaddition to parvoviral viral DNA. The recombinant vector genome willtypically include at least one targeting construct.

The term “replicating cell” refers to a cell that is passing through thecell cycle, including the S and M phases of DNA synthesis and mitosis.

The term “subsequence” in the context of a particular nucleic acidsequence refers to a region of the nucleic acid equal to or smaller thanthe specified nucleic acid.

A “target locus,” as used herein, refers to a region of a cellulargenome at which a genetic modification is desired. The target locustypically includes the specific nucleotides to be modified, as well asadditional nucleotides on one or both sides of the modification sites.

A “targeting construct” refers to a DNA molecule that is present in therecombinant parvoviral vectors used in the methods of the invention andincludes a region that is identical to, or substantially identical to, aregion of the target locus, except for the modification or modificationsthat are to be introduced into the host cell genome at the target locus.The modification can be at either end of the targeting construct, or canbe internal to the targeting construct. The modification can be one ormore deletions, point mutations, and/or insertions, or combinationsthereof, compared to DNA in the wild-type target cell.

The term “transduction” refers to the transfer of genetic material byinfection of a recipient cell by a recombinant viral vector.

A cell that has received recombinant parvoviral vector DNA, therebyundergoing genetic modification, is referred to herein as a “transducedcell,” a “transfected cell,” a “modified cell,” or a “recombinant cell,”as are progeny and other descendants of such cells.

The term “transgenic cell” refers to a cell that includes a specificmodification of the cell's chromosomal or other nucleic acids, whichspecific modification was introduced into the cell, or an ancestor ofthe cell. Such modifications can include one or more point mutations,deletions, insertions, or combinations thereof. When referring to ananimal, the term “transgenic” means that the animal includes cells thatare transgenic. An animal that is composed of both transgenic cells andnon-transgenic cells is referred to herein as a “chimeric” animal.

The term “vector” refers to an agent for transferring a nucleic acid (ornucleic acids) to a host cell. A vector comprises a nucleic acid thatincludes the nucleic acid fragment to be transferred, and optionallycomprises a viral capsid or other materials for facilitating entry ofthe nucleic acid into the host cell and/or replication of the vector inthe host cell (e.g., reverse transcriptase or other enzymes which arepackaged within the capsid, or as part of the capsid).

The term “viral vector” refers to a vector that comprises a viralnucleic acid and can also include a viral capsid and/or replicationfunctions.

Description of the Preferred Embodiments

The present invention provides methods of producing a vertebrate cellthat has a specific modification of a target locus. Genetically modifiedcells and animals produced using these methods are also provided. Themethods involve introducing into the cell a recombinant parvoviralvector that is capable of targeting a genetic modification to aparticular target locus by homologous pairing. The recombinant viralgenomes of the parvoviral vectors used in the methods include atargeting construct that includes a DNA sequence that is substantiallyidentical to the target locus except for the modification beingintroduced. Upon introduction of the recombinant viral genome into thecell, homologous pairing occurs between the targeting construct and thetarget locus, resulting in the introduction of the specific geneticmodifications into the target locus.

The methods of the invention make possible precise modifications of thegenome of a cell. This allows one to avoid undesired effects, such asdisruption of a desirable gene by insertion of an exogenous gene, thatcan occur when other methods of modifying a genome are used. Moreover,one can achieve precise changes in a gene or a control region, forexample, making possible the correction of an endogenous gene withouthaving to insert a correct copy of the gene elsewhere in the genome. Themethods avoid the frequently observed “position effect” in which thelevel of expression of an exogenous gene is highly dependent upon thelocation in a cell's genomic DNA at which the exogenous gene becomesintegrated. The methods also make possible the modification of genesthat are too large to be introduced into cells by other methods. Ratherthan having to introduce an entire copy of the gene that includes thedesired modifications, one can use the methods of the invention tomodify only a desired portion of the gene.

The methods of the invention use recombinant parvoviral vectors toinsert DNA that includes desired genetic modifications into thevertebrate cells to be modified. A general introduction to humanparvoviruses is found, e.g., in Pattison (1994) Principles and Practiceof Clinical Virology (Chapter 23) Zuckerman et al. eds, John Wiley &Sons Ltd., and also in Berns (1991) “Parvoviridae and theirReplication,” In Fundamental Virology, Fields, Ed., Raven Press, NewYork, pp. 817-837, as well as references cited in each. The bestcharacterized of the human parvoviruses are B19 and AAV, both of whichare used as the basis for cell transduction vectors, e.g., for genetherapy. Other parvoviral vectors that can be used include, but are notlimited to, the parvoviral vectors LuIII (Maxwell et al. (1993) HumanGene Ther. 4: 441-450) and minute virus of mice (mvm) (Russell et al.(1992) J. Virol. 66: 2821-2828.

In a preferred embodiment, the methods use a viral vector derived froman adeno-associated virus (AAV). AAVs are single-stranded,replication-defective DNA viruses with a 4.7 kb genome. Adeno-associatedviruses are readily obtained, and their use as vectors for gene deliverywas described in, for example, Muzyczka (1992) Curr. Top. Microbiol.Immunol. 158: 97-129, U.S. Pat. No. 4,797,368, and PCT Application WO91/18088. Samulslci (1993) Current Opinion in Genetic and Development 3:74-80 and the references cited therein provides an overview of the AAVlife cycle. For a general review of AAVs and of the adenovirus or herpeshelper functions see, Berns and Bohensky (1987) Advances in VirusResearch, Academic Press., 32: 243-306. The genome of AAV is describedin Srivastava et al. (1983) J. Virol., 45: 555-564. Carter et al., U.S.Pat. No. 4,797,368, describe many of the relevant design considerationsfor constructing recombinant AAV vectors. See also, Carter WO 93/24641.Additional references describing AAV vectors include, for example, Westet al. (1987) Virology 160: 38-47; Kotin (1994) Human Gene Therapy5:793-801; and Muzyczka (1994) J. Clin. Invest. 94: 1351. Constructionof recombinant AAV vectors is also described in a number of additionalpublications, including Lebkowski, U.S. Pat. No. 5,173,414; Lebkowski etal. (1988) Mol. Cell. Biol. 8: 3988-3996; Tratschin et al. (1985) Mol.Cell. Biol. 5(11):3251 3260; Tratschin et al. (1984) Mol. Cell. Biol.,4: 2072-2081; Hermonat and Muzyczka (1984) Proc. Nat'l. Acad. Sci. USA,81: 6466-6470; McLaughlin et al. (1988) and Samulski et al. (1989) J.Virol., 63: 03822-3828. AAV is a defective human parvovirus, meaningthat the virus is capable of replicating and forming virus particlesonly in cells that are also infected with a helper virus. To obtainintegration of an AAV genome into a mammalian cell, the cell is infectedwith the AAV in the absence of a helper virus.

Parvoviral genomes have an inverted terminal repeat sequence (ITR) ateach end. For use in the methods of the invention, the recombinantparvoviral vector genomes will typically have all or a portion of atleast one of the ITRs or a functional equivalent, which is generallyrequired for the parvoviral vectors to replicate and be packaged intoparvovirus particles. A functional equivalent of an ITR is typically aninverted repeat which can form a hairpin structure. Both ITRs are oftenpresent in the recombinant parvoviral vector DNAs used in the methods.One can use the viral genomes in either single-stranded ordouble-stranded form.

The recombinant viral genomes of the parvoviral vectors used in themethods for genetically modifying vertebrate cells will include atargeting construct that, except for the desired modification, isidentical to, or substantially identical to, the target locus at whichgenetic modification is desired. The targeting construct will generallyinclude at least about 20 nucleotides, preferably at least about 100,and more preferably about 1000-5000 nucleotides or more, that areidentical to, or substantially identical to, the nucleotide sequence ofa corresponding region of the target locus. By “substantially identical”is meant that this portion of the targeting construct is at least about80% identical; more preferably, at least about 90%, and most preferablyat least about 99% identical to the corresponding region of the targetlocus.

The targeting construct will also include the genetic modification ormodifications that are to be introduced into the target locus. Themodifications can include one or more insertions, deletions, or pointmutations, or combinations thereof, relative to the DNA sequence of thetarget locus. For example, to modify a target locus by introducing apoint mutation, the targeting construct will include a DNA sequence thatis at least substantially identical to the target locus except for thespecific point mutation to be introduced. Upon introduction of therecombinant viral genome into the cell, homologous pairing occursbetween the portions of the targeting construct that are substantiallyidentical to the corresponding regions of the target locus, after whichthe DNA sequence of the mutation to be introduced that is present in thetargeting construct replaces that of the target locus.

A targeting construct can have the genetic modifications at either endof, or within the region of the targeting construct that is identicalto, or substantially identical to, the target locus. To delete a portionof a target locus, for example, the genetic modification will generallybe within the targeting construct, being flanked by two regions ofsubstantial identity to the target locus. Homologous pairing between thetwo regions of substantial identity and their corresponding regions ofthe target locus result in a portion of the sequence of the targetingconstruct, including the deletion, becoming incorporated into the targetlocus. Deletions can be precisely targeted to a desired location by thismethod. Similarly, genetic modifications that involve site-specificinsertion of DNA sequences into the target locus can be made by use of atargeting construct that has the DNA sequence to be inserted flanked byor next to regions of substantial identity to the target locus.Homologous pairing between the targeting construct and the correspondingregions of the target locus is followed by incorporation of theinsertion sequence into the target locus.

The methods of the invention can be used to introduce modifications atmore than one target locus. For example, to introduce one or moremodifications at a second target locus in a cellular genome, the cellcan be contacted with a parvoviral vector that has a recombinant viralgenome that has a targeting construct that is at least substantiallyidentical to the second target locus, except for the desiredmodification or modifications. The targeting construct for the secondtarget locus can be present in the same parvoviral vector as thetargeting construct for the first target locus, or can be present in asecond parvoviral vector. Where the first and second targetingconstructs are present in different parvoviral vectors, the cells can betransduced with the vectors either sequentially or simultaneously. Toobtain modifications at more than two target loci, this process issimply repeated as desired.

Structural genes, regulatory regions, and other sequences within thegenomic or other DNA of a vertebrate cell are amenable to modificationusing the methods of the invention. For example, one can introducespecific changes within structural genes that can alter the gene productof the gene, or prevent the gene product from being expressed. A“structural gene” refers to the transcribed region of a gene, whether ornot the gene is transcribed in a particular cell. In this embodiment,the recombinant viral genome can include a targeting construct that isidentical to, or substantially identical to, the target locus, with theexception of the specific nucleotide changes to be introduced.Homologous pairing between the targeting construct and the target locusin the cellular DNA results in the modifications present in thetargeting construct becoming incorporated into the target locus. Wherethe gene product is a polypeptide, for example, one can use the methodsof the invention to obtain a gene that encodes a polypeptide having oneor more specific amino acid substitutions, insertions, or deletionscompared to the polypeptide encoded by the native gene. The methodsallow one to replace a codon that specifies an amino acid that, whenpresent, results in the polypeptide being inactive, or less active thandesired, with a codon specifies an amino acid that restores normalactivity to the polypeptide. Many genetic diseases that arecharacterized by one or more mutations that result in amino acid changesare correctable using the methods of the invention. As another example,a target region can be modified by substituting a codon that specifies aglycosylation site for a codon that encodes an amino acid that is notpart of a glycosylation site, or vice versa. A protease cleavage sitecan be created or destroyed, as yet another example. A nonsense codonpresent in the target locus can be changed to a sense codon, or wheredisruption of the polypeptide is desired, one can introduce a nonsensemutation into the target locus. One can obtain a fusion protein byincorporating into the targeting construct an exogenous DNA that codesfor the portion of the fusion protein that is to be joined to anendogenous protein; the exogenous DNA will be in the proper readingframe for translation of the fusion protein upon incorporation of theDNA sequence of the targeting construct into the cellular genome at thetarget locus.

Similarly, where the gene product is a nucleic acid, the methods can beused for modification of the gene products. RNA genes that can bemodified using the methods of the invention include those from which areexpressed tRNAs, ribosomal RNAs, ribozymes, telomerase subunits, and thelike. Alternatively, the methods can be used to construct a gene forwhich the gene product consists of an endogenous nucleic acid linked toan exogenous nucleic acid. For example, an exogenous DNA that whentranscribed produces a catalytic RNA can be linked to an endogenousgene. The RNA that is transcribed from this fusion gene can hybridize toendogenous nucleic acids that are substantially complementary to theendogenous portion of the fusion gene, after which the portion of thehybrid ribozyme that is expressed from the exogenous DNA can catalyzeits usual reaction. Thus, the fusion gene obtained using the methods ofthe invention provides a means for targeting a ribozyme.

The methods of the invention also are useful for substituting, deletingor inserting nucleotides that make up regulatory regions that areinvolved in expressing a gene of interest. The altered regulatory regioncan change the expression of the gene by, for example, increasing ordecreasing the level of expression of the gene compared to the level ofexpression under equivalent conditions in an unmodified cell. Themodifications can, for example, result in expression of the gene undersituations where the gene would not typically be expressed, or canprevent expression of a gene that normally would be expressed underparticular circumstances. One can use the methods to insert aheterologous transcription control element, or modify an endogenouscontrol element, such as a promoter, enhancer, transcription terminationsignal, at a location relative to the gene of interest that isappropriate for influencing expression of the gene. By replacing aconstitutive promoter with an inducible promoter, for instance, one cantie expression of the gene to the presence or absence of a particularenvironmental or developmental stimulus. Similarly, regions that areinvolved in post-transcriptional modification, such as RNA splicing,polyadenylation, translation, as well as regions that code for aminoacid sequences involved in post-translational modification, can beinserted, deleted, or modified. Examples of gene expression controlelements that can be modified or replaced using the methods include, butare not limited to, response elements, promoters, enhancers, locuscontrol regions, binding sites for transcription factors and otherproteins, other transcription initiation signals, transcriptionelongation signals, introns, RNA stability sequences, transcriptiontermination signals, polyadenylation sites, and splice sites. Expressionof a gene can also be modulated by using the methods of the invention tointroduce or destroy DNA methylation sites.

In one embodiment, the methods of the invention are used to obtainselective expression of a nucleic acid in a cell. Selective expressionof a nucleic acid refers to the ability of the nucleic acid to beexpressed in a desired cell type and/or under desired conditions (e.g.,upon induction) but not to be substantially expressed in undesired celltypes and/or under undesired conditions. Thus, the site and degree ofexpression of a particular nucleic acid sequence is regulated in adesired fashion. This is accomplished by, for example, introducingsite-specific nucleotide substitutions, deletions, or insertions tocreate a nucleotide sequence that comprises a control element that isselectively expressed in the desired cell type and/or under desiredconditions. This can be accomplished entirely by changing nucleotidesthat are already present in the target locus, or by incorporating intothe target locus an exogenous DNA that includes a sequence thatfunctions as all or part of a control element, or by a combination ofthese modifications.

For example, one can use the methods of the invention to introduce ordisrupt a response element, which is a cis-acting nucleic acid sequencethat interacts with a trans-activating or trans-repressing compound(usually a protein or a protein complexed with another material) torespectively stimulate or suppress transcription. Response elements thatcan be introduced or eliminated using the methods of the inventioninclude cell-selective response elements, hormone receptor responseelements, carbohydrate response elements, antibiotic response elements,and the like. A cell-selective response element is capable of beingactivated by a trans-activating regulatory element that is selectivelyproduced in the cell type(s) of interest. The choice of cell-selectiveresponse element used in the methods depends upon whether the cell inwhich induction or repression of expression is desired produces thetrans-activator that acts on the response element. For example,selective expression of a gene in pancreatic acinar cells, lens tissue,B cells, liver cells, and HIV-infected cells can be achieved by usingthe methods of the invention to introduce, respectively, an elastase Ienhancer, a gamma crystallin gene response elements, an immunoglobulinheavy and/or light chain enhancer, a liver enhancer such as anα-1-antitrypsin or serum albumin enhancer, a chorionic gonadotropinα-chain or β-chain enhancer, an interleukin-2 (IL-2) enhancer, an IL-2receptor enhancer, or a human immunodeficiency virus (HIV) responseelement such as the TAR site.

Hormone receptor response elements, which can be activated or repressedwhen a hormone, or a functional equivalent thereof, interacts with acellular receptor for that hormone, can be introduced into a desiredlocation using the methods of the invention. The hormone-receptorcomplex is internalized by the cell, where it selectively interacts withthe appropriate hormone receptor response element (either directly orindirectly), thereby activating or repressing expression of genesoperatively linked to the element. To obtain hormone-responsiveinduction or repression of expression, the methods are used to create ahormone response element upstream of a gene to be regulated. Expressionof the gene will be regulated by the hormone in those cells that expressreceptors for the given hormone.

An antibiotic response element is regulated by the presence or absenceof an antibiotic. For example, a tetracycline response element isresponsive to tetracycline. Similarly, a carbohydrate response elementis regulated by the presence or absence of certain carbohydrates oranalogs thereof. Other response elements, as well as promoters,enhancers, and other regulatory regions, are well known to those ofskill in the art. These can also be created or destroyed by use of themethods of the invention.

The methods of the invention can also be used to modify nucleic acidsequences that are involved in other cellular processes such as DNAreplication (see, e.g., Kornberg and Baker, DNA Replication, 2^(nd) Ed.,WH Freeman & Co., 1991), as well as matrix attached regions (see, e.g.,Bode et al. (1996) Crit. Rev. Eukaryot. Gene. Expr. 6: 115-38; Boulikas(1993) J. Cell. Biochem. 52: 14-22), chromatin recombination hotspots(see, e.g., Smith (1994) Experientia 50: 234-41), and the like.

The invention also provides methods of introducing a recombinationsignal into a cell. In preferred embodiments, a specific recombinaseenzyme is available which can catalyze recombination at therecombination signal. To introduce a recombination signal into acellular genome, one or more recombination signals is included in thetargeting construct, flanked by polynucleotide sequences that are atleast substantially identical to the target locus. Homologous pairingfollowed by gene repair results in incorporation of the recombinationsignal(s) into the target locus.

One suitable recombination system is the Cre-lox system. In the Cre-loxsystem, the recombination sites are referred to as “lox sites” and therecombinase is referred to as “Cre.” When lox sites are in parallelorientation (i.e., in the same direction), then Cre catalyzes a deletionof the polynucleotide sequence between the lox sites. When lox sites arein the opposite orientation, the Cre recombinase catalyzes an inversionof the intervening polynucleotide sequence. Thus, for example, one coulduse the methods of the invention to introduce two lox sites into targetlocus, oriented in opposite directions, and obtain inversion of theregion between the lox sites by contacting the lox sites with the Crepolypeptide. If the two lox sites flank a promoter, for example, onecould turn expression of a gene on or off simply by controlling thepresence or absence of the Cre polypeptide. Such sites are also usefulfor introducing DNA that also includes a recombination signal at thelocation of the recombination signal in the target locus. In someembodiments, a gene encoding the Cre polypeptide is present in the cell,under the control of either a constitutive or an inducible promoter.

Several different lox sites are known, including lox511 (Hoess R. etal., Nucleic Acids Res. 14:2287-2300 (1986)), lox66, lox71, lox76,lox75, lox43, lox44 (Albert H. et al., Plant J. 7(4): 649-659 (1995)).This system works in various host cells, including mammalian cells (U.S.Pat. No. 4,959,317; Sauer, B. et al., Proc. Nat'l. Acad. Sci. USA85:5166-5170 (1988); Sauer, B. et al., Nucleic Acids Res. 17:147-161(1989)); Saccharomyces cerevisiae (Sauer, B., Mol Cell Biol. 7:2087-2096(1987)); and plants such as tobacco (Dale, E. et al., Gene 91:79-85(1990)) and Arabdiopsis (Osborne, B. et al., Plant J. 7(4):687-701(1995)) Use of the Cre-lox system in plants is also described in U.S.Pat. No. 5,527,695 and PCT application No. WO 93/0128.

Several other recombination systems are also suitable for use in theinvention. These include, for example, the FLP/FRT system of yeast(Lyznik, L. A. et al., Nucleic Acids Res. 24(19):3784-9 (1996)), the Ginrecombinase of phage Mu (Crisona, N. J. et al., J. Mol. Biol.243(3):437-57 (1994)), the Pin recombinase of E. coli (see, e.g.,Kutsukake K, et al., Gene 34(2-3):343-50 (1985)), the PinB, PinD andPinF from Shigella (Tominaga A et al., J. Bacteriol. 173(13):4079-87(1991)), and the R/RS system of the pSRi plasmid (Araki, H. et al., J.Mol. Biol. 225(1):25-37 (1992)). Thus, recombinase systems are availablefrom a large and increasing number of sources.

Through their use of parvoviral vectors to deliver the recombinant viralgenome to a cell, the methods of the invention result in desiredspecific genetic modification events occurring at a much higherfrequency than previously possible with other methods of site-specificmodification of DNA in vertebrate cells. Desired modificationfrequencies of greater than 0.01% or greater are typically obtainedusing the methods; indeed, efficiencies greater than 0.1%, and evengreater than 1% can be obtained using the methods. The efficiency ofgenetic modification depends in part on the multiplicity of infection(MOI; defined herein in units of vector particles per cell) used for thetransduction, as well as the type of cell being transduced. In a typicalembodiment, a MOI of about 1 to 10¹² is used to transduce a cellobtained from a continuous cell line; more preferably the MOI is atleast about 10⁴, and most preferably the MOI used in the methods of theinvention is at least about 10⁶ vector particles per cell.

The methods are useful for introducing genetic modifications into anycells that are susceptible to transduction by the recombinant parvoviralvectors. Such cells can be obtained from many vertebrate species,including mammals, birds, reptiles, amphibians, fish, and the like. Forexample, cells from mammals such as human, cow, pig, goat, sheep,rodent, and the like can be modified using these methods. Cells that canbe modified using the methods of the invention include brain, muscle,liver, lung, bone marrow, heart, neuron, gastrointestinal, kidney,spleen, and the like. Also amenable to genetic modification using themethods are germ cells, including ovum and sperm, fertilized egg cells,embryonic stem cells, and other cells that are capable of developinginto an organism, or a part of an organism such as an organ. Forexample, one can use the methods of the invention to modify a cell thatis to be a nucleus donor in a nuclear transplantation.

Both primary cells (also referred to herein as “normal cells”) and cellsobtained from a cell line are amenable to modification using the methodsof the invention. Primary cells include cells that are obtained directlyfrom an organism or that are present within an organism, and cells thatare obtained from these sources and grown in culture, but are notcapable of continuous (e.g., many generations) growth in culture. Forexample, primary fibroblast cells are considered primary cells. Themethods are also useful for modifying the genomes of cells obtained fromcontinuous, or immortalized, cell lines, including, for example, tumorcells and the like, as well as tumor cells obtained from organisms.Cells can be modified in vitro, ex vivo, or in vivo using the methodsand vectors of the invention.

The methods are useful for modifying the genomes of vertebrate cellorganelles, as well as nuclear genomes. For example, one can use themethods of the invention to modify a target locus in the mitochondrialgenome of a cell by including in the recombinant viral genome atargeting construct that, except for the desired modification ormodifications, is at least substantially identical to a target locus inthe mitochondrial genome.

A. Preparing Vectors

The practice of this invention involves the construction of recombinantparvoviral vector genomes and, optionally, the packaging of these viralgenomes into viral particles. Methods for achieving these ends are knownin the art. A wide variety of cloning and in vitro amplification methodssuitable for the construction of recombinant viral genomes arewell-known to persons of skill Examples of these techniques andinstructions sufficient to direct persons of skill through many cloningexercises are found in Berger and Kimmel, Guide to Molecular CloningTechniques, Methods in Enzymology 152 Academic Press, Inc., San Diego,Calif. (Berger); Sambrook et al. (1989) Molecular Cloning—A LaboratoryManual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold SpringHarbor Press, NY, (Sambrook); Current Protocols in Molecular Biology, F.M. Ausubel et al., eds., Current Protocols, a joint venture betweenGreene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1994Supplement) (Ausubel); Cashion et al., U.S. Pat. No. 5,017,478; andCarr, European Patent No. 0,246,864.

Examples of techniques sufficient to direct persons of skill through invitro amplification methods are found in Berger, Sambrook, and Ausubel,as well as Mullis et al. (1987) U.S. Pat. No. 4,683,202; PCR Protocols AGuide to Methods and Applications (Innis et al. eds) Academic Press Inc.San Diego, Calif. (1990) (Innis); Arnheim & Levinson (Oct. 1, 1990) C&EN36-47; The Journal Of NIH Research (1991) 3: 81-94; Kwoh et al. (1989)Proc. Natl. Acad. Sci. USA 86: 1173; Guatelli et al. (1990) Proc. Natl.Acad. Sci. USA 87: 1874; Lomell et al. (1989) J. Clin. Chem. 35: 1826;Landegren et al. (1988) Science 241: 1077-1080; Van Brunt (1990)Biotechnology 8: 291-294; Wu and Wallace (1989) Gene 4, 560; andBarringer et al. (1990) Gene 89: 117. Oligonucleotide synthesis, usefulin cloning or amplifying nucleic acids, is typically carried out oncommercially available solid phase oligonucleotide synthesis machines(Needham-VanDevanter et al. (1984) Nucleic Acids Res. 12:6159-6168) orchemically synthesized using the solid phase phosphoramidite triestermethod described by Beaucage et al. ((1981) Tetrahedron Letts. 22 (20):1859-1862.

Typically, the recombinant viral genomes are initially constructed asplasmids using standard cloning techniques. The targeting constructs areinserted into the viral vectors, which include at least one of the twoinverted terminal repeats or their functional equivalent. In someembodiments, the viral vector DNA is packaged into virions for use toinfect the target cells. Viral vectors to be packaged can include in theviral genome DNA sequences necessary for replication and packaging ofthe recombinant viral genome into virions. In most embodiments, however,one or more of the replication and/or packaging polypeptides is providedby a producer cell line and/or a helper virus (e.g., adenovirus orherpesvirus). These helper functions include, for example, the Repexpression products, which are required for replicating the AAV genome(see, e.g., Muzyczka, N. (1992) Current Topics in Microbiol. andImmunol. 158: 97-129 and Kotin, R. M. (1994) Human Gene Therapy5:793-801). The human herpesvirus 6 (HHV-6) rep gene can serve as asubstitute for an AAV rep gene (Thomson et al. (1994) Virology 204:304-311). The cap region, which encodes the capsid proteins VP1, VP2,and VP3, or functional homologues thereof, is also typically provided bya helper virus or producer cell line (Id.).

The recombinant viral genomes are grown as a plasmid and packaged intovirions by standard methods. See, e.g., Muzyczka, supra., Russell et al.(1994) Proc. Nat'l. Acad. Sci. USA 91: 8915-8919, Alexander et al.(1996) Human Gene Ther. 7: 841-850; Koeberl et al. (1997) Proc. Nat'l.Acad. Sci. USA 94: 1426-1431; Samulski et al. (1989) J. Virol. 63:3822-3828; Tratschin et al. (1985) Mol. Cell. Biol. 5: 3251-3260; andHermonat and Muzyczka (1984) Proc. Nat'l. Acad. Sci. USA 81: 6466-6470.

The recombinant viral genomes can be introduced into target cells by anyof several methods. For example, as discussed above, one can package theviral genomes into parvoviral virions which are then used to infect thetarget cells. Alternatively, the recombinant viral genomes can beintroduced into cells in an unpackaged form. For example, standardmethods for introducing DNA into cells can be employed to introduce theviral genomes, such as by microinjection, transfection, electroporation,lipofection, lipid encapsulation, biolistics, and the like. Therecombinant viral genomes can be incorporated into viruses other thanparvoviruses (e.g., an inactivated adenovirus), or can be conjugated toother moieties for which a target cell has a receptor and/or a mechanismfor cellular uptake (see, e.g., Gao et al. (1993) Hum. Gene Ther. 4:17-24). The recombinant viral genomes can be introduced into either thenucleus or the cytoplasm of the target cells.

Methods of transfecting and expressing genes in vertebrate cells areknown in the art. Transducing cells with viral vectors can involve, forexample, incubating vectors with cells within the viral host range underconditions and concentrations necessary to cause transduction. See,e.g., Methods in Enzymology, vol. 185, Academic Press, Inc., San Diego,Calif. (D. V. Goeddel, ed.) (1990) or M. Krieger, Gene Transfer andExpression—A Laboratory Manual, Stockton Press, New York, N.Y.; andMuzyczka (1992) Curr. Top. Microbiol. Immunol. 158: 97-129, andreferences cited in each. The culture of cells, including cell lines andcultured cells from tissue samples is well known in the art. Freshney(Culture of Animal Cells, a Manual of Basic Technique, Third editionWiley-Liss, New York (1994)) provides a general guide to the culture ofcells.

The recombinant viral genomes and/or other components of a recombinantviral vector can be manipulated to improve targeting efficiency. Thesetargeting enhancers can include, for example, adducts, pyrimidinedimers, and/or other DNA alterations that can induce cellular DNAsynthesis, repair, and/or recombination systems, that are introducedinto the viral genomes. Such alterations can include, modification ofnucleotides in the viral DNA, such as elimination of one or more sugars,bases, and the like. For example, the parvoviral vectors can be treatedwith DNA damaging agents such as UV light, gamma irradiation, andalkylating agents. The modifications can be performed on the viral DNAin vitro or during or after packaging of the viral DNA into virions.

Other targeting enhancers that can be included are recombinogenicproteins. See, e.g., Pati et al. (1996) Molecular Biol. of Cancer 1:1;Sena and Zarling (1996) Nature Genet. 3: 365; Revet et al. (1993) J.Mol. Biol. 232: 779-791; Kowalczkowski & Zarling in Gene Targeting (CRC1995, Ch. 7). The parvoviral vector nucleic acids can be associated withthe recombinogenic proteins prior to being introduced into the cells, orthe recombinogenic proteins can be introduced into the cellsindependently of the parvoviral vectors. In one presently preferredembodiment, the parvoviral vector is packaged in the presence of therecombinogenic protein, resulting in recombinogenic protein becomingpackaged into the viral particles. The best-characterized recombinogenicprotein is recA from E. coli and is available from Pharmacia (PiscatawayN.J.). In addition to the wild-type protein, a number of mutantrecA-like proteins have been identified (e.g., recA803). Further, manyorganisms have recA-like recombinases (e.g., Ogawa et al. (1993) ColdSpring Harbor Symp. Quant. Biol. 18: 567-576; Johnson and Symington(1995) Mol. Cell. Biol. 15: 4843-4850; Fugisawa et al. (1985) Nucl.Acids Res. 13: 7473; Hsieh et al. (1986) Cell 44: 885; Hsieh et al.(1989) J. Biol. Chem. 264: 5089; Fishel et al. (1988) Proc. Nat'l. Acad.Sci. USA 85: 3683; Cassuto et al. (1987) Mol. Gen. Genet. 208: 10; Ganeaet al. (1987) Mol. Cell. Biol. 7: 3124; Moore et al. (1990) J. Biol.Chem. 19: 11108; Keene et al. (1984) Nucl. Acids Res. 12: 3057; Kimiec(1984) Cold Spring Harbor Symp. Quant. Biol. 48: 675; Kimeic (1986) Cell44: 545; Kolodner et al. (1987) Proc. Nat'l. Acad. Sci. USA 84: 5560;Sugino et al. (1985) Proc. Nat'l. Acad. Sci. USA 85: 3683; Halbrook etal. (1989) J. Biol. Chem. 264: 21403; Eisen et al. (1988) Proc. Nat'l.Acad. Sci. USA 85: 25 7481; McCarthy et al. (1988) Proc. Nat'l. Acad.Sci. USA 85: 5854; Lowenhaupt et al. (1989) J. Biol. Chem. 264: 20568.Examples of such recombinase proteins include, for example, recA,recA803, uvsX (Roca (1990) Crit. Rev. Biochem. Molec. Biol. 25: 415),sepl (Kolodner et al. (1987) Proc. Nat'l. Acad. Sci. USA 84: 5560;Tishkoff et al., Mol. Cell. Biol. 11: 2593), RuvC (Dunderdale et al.(1991) Nature 354: 506), DST2, KEMI, XRAU (Dykstra et al. (1991) Mol.Cell. Biol. 11: 2583), STPα/DST1 (Clark et al. (1991) Mol. Cell. Biol.11: 2576), HPP-1 (Moore et al. (1991) Proc. Nat'l. Acad. Sci. USA 88:9067), and other eukaryotic recombinases (Bishop et al. (1992) Cell 69:439; Shinohara et al., Cell 69: 457). See also, PCT patent applicationPCT/US98/000852 (WO 98/31837).

The efficiency of gene targeting can also be improved by treating thehost cell in conjunction with the introduction of the recombinant viralgenome. For example, one can administer to the target cells an agentthat affects the cell cycle. These agents include, for example, DNAsynthesis inhibitors (e.g., hydroxyurea, aphidicolin), microtubuleinhibitors (e.g., vincristine), and genotoxic agents (e.g., radiation,alkylators). Other agents that can improve the efficiency of genetargeting include those that affect DNA repair, DNA recombination, DNAsynthesis, protein synthesis, and levels of receptors for AAV. Also ofinterest are agents that affect, chromatin packaging, gene silencing,DNA methylation, and the like, as less condensed DNA is more likely tobe accessible for gene targeting. These agents include, for example,topoisomerase inhibitors such as Etoposide and camptothecin, and histonedeacetylase inhibitors such as sodium butyrate and trichostatin A.Agents that inhibit apoptosis can also increase gene targeting by virtueof their ability to reduce the tendency of high concentrations of AAV toinduce apoptosis. Suitable agents for these applications are describedin, for example, U.S. Pat. No. 5,604,090, Russell et al. (1995) Proc.Nat'l. Acad. Sci. USA 92: 5719; Chen et al. (1997) Proc. Nat'l. Acad.Sci. USA 94: 5798; Alexander et al. (1994) J. Virol. 68: 8282; andFerrari et al. ((1995) J. Neurosci. 15: 2857-66, (1998) Mol. Cell. Biol.18: 6482-92, (1994) EMBO J. 13: 5922-8 (70:3227)).

The parvoviral vectors can also be targeted to a particular cell ortissue type by use of a virion or delivery vehicle that displays amolecule that binds to a moiety that is specific for a desired targetcell. For example, an antibody that binds to a polypeptide found oncancer cells can be displayed on the delivery vehicle. In someembodiments, polynucleotides that encode a polypeptide which canspecifically bind to the target cell are incorporated into a gene thatencodes a parvoviral capsid protein. Upon packaging of the parvoviralvector genome, the modified capsid is displayed on the surface of thevirion, thus allowing the virions to preferentially deliver the nucleicacid to the desired target cell. Modification of parvoviral capsidproteins for other purposes, as well as cell lines useful for expressingthe genes that encode the modified proteins and methods of in vitropackaging using the modified capsid proteins, are described in U.S. Pat.No. 5,863,541, issued Jan. 26, 1999.

One can also improve gene targeting by using only one strand, eitherplus or minus, of the recombinant viral genomes. For example, use of apopulation of parvoviral vectors that each carry a plus strand, or thateach carry a minus strand, can increase the efficiency of genetargeting. Alternatively, one can use a combination of plus and minusstrands, each delivered by a different vector.

B. Identification of Cells Having Genetic Modifications

Because of the high frequencies with which specific geneticmodifications occur using the methods of the invention, selection orscreening for individual cells that include the desired modification isnot necessary for many uses. Where it is desirable to identify cellsthat have incorporated a desired genetic modification, one can usetechniques that are well known to those of skill in the art. Forexample, PCR and related methods (such as ligase chain reaction) areroutinely used to detect specific changes in nucleic acids (see, Innis,supra, for a general description of PCR techniques). Hybridizationanalysis under conditions of appropriate stringency are also suitablefor detecting specific genetic modifications. Many assay formats areappropriate, including those reviewed in Tijssen (1993) LaboratoryTechniques in Biochemistry and Molecular Biology—Hybridization withNucleic Acid Probes, Parts I and II, Elsevier, New York; and Choo (ed)(1994) Methods In Molecular Biology Volume 33—In Situ HybridizationProtocols, Humana Press Inc., New Jersey (see also, other books in theMethods in Molecular Biology series). A variety of automated solid-phasedetection techniques are also appropriate. For instance, very largescale immobilized polymer arrays (VLSIPS™) are used for the detection ofspecific mutations in nucleic acids. See, Tijssen (supra), Fodor et al.(1991) Science, 251: 767-777 and Sheldon et al. (1993) ClinicalChemistry 39(4): 718-719.

These methods can be used to detect the specific genetic modificationsthemselves, or can be used to detect changes that result from themodification. For example, one can use hybridization or other methods todetect the presence or absence of a particular mRNA in a cell that has amodification in the promoter region.

One can also detect changes in the phenotype of the cells by othermethods. For example, where a genetic modification results in apolypeptide being expressed in modified cells under conditions that anunmodified cell would not express the polypeptide, or vice versa,antibodies against the polypeptide can be used to detect expression.When the modified cells are in a vertebrate, the antibodies can be usedto detect the presence or absence of the protein in the bloodstream orother tissue, for example. Where the genetic modification changes thestructure of a polypeptide, one can obtain an antibody that recognizesthe unmodified polypeptide but not the modified version, or vice versa.Methods of producing polyclonal and monoclonal antibodies are known tothose of skill in the art, and many antibodies are available. See, e.g.,Coligan (1991) Current Protocols in Immunology Wiley/Greene, N.Y.; andHarlow and Lane (1989) Antibodies: A Laboratory Manual, Cold SpringHarbor Press, NY; Stites et al. (eds.) Basic and Clinical Immunology(4th ed.) Lange Medical Publications, Los Altos, Calif., and referencescited therein; Goding (1986) Monoclonal Antibodies: Principles andPractice (2d ed.) Academic Press, New York, N.Y.; and Kohler andMilstein (1975) Nature 256: 495-497. Other suitable techniques forantibody preparation include selection of libraries of recombinantantibodies in phage or similar vectors. See, Huse et al. (1989) Science246: 1275-1281 and Ward et al. (1989) Nature 341: 544-546. Vaughan etal. (1996) Nature Biotechnology, 14: 309-314 describe human antibodieswith subnanomolar affinities isolated from a large 20 non-immunizedphage display library. Chhabinath et al. describe a knowledge-basedautomated approach for antibody structure modeling ((1996) NatureBiotechnology 14: 323-328). Specific monoclonal and polyclonalantibodies and antisera will usually bind to their corresponding antigenwith a K_(D) of at least about 0.1 mM, more usually at least about 1 μM,preferably at least about 0.1 μM or better, and most typically andpreferably, 0.01 μM or better. One can also detect the enzymaticactivity (or loss thereof) of the modified enzyme.

Genetically modified cells can also be identified by use of a selectableor screenable marker that is incorporated into the cellular genome. Aselectable marker can be a gene that codes for a protein necessary forthe survival or growth of the cell, so only those host cells thatcontain the marker are capable of growth under selective conditions. Forexample, where the methods of the invention are used to introduce agenetic modification that places a gene that is required for cell growthunder the control of an inducible promoter, cells that have incorporatedthe desired modification can be selected by growing the cells underselective conditions that also induce expression of the gene. Typicalselection genes encode proteins that (a) confer resistance toantibiotics or other toxic substances, e.g., ganciclovir, neomycin,hygromycin, G418, methotrexate, etc.; (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media. The choice of the proper selectable marker will depend onthe host cell, and appropriate markers for different hosts are wellknown in the art. A screenable marker is a gene that codes for a proteinwhose activity is easily detected, allowing cells expressing such amarker to be readily identified. Such markers include, for example,β-galactosidase, β-glucuronidase, and luciferase. Other markers includethose that are identifiable by fluorescence-activated cell sorting. Forexample, a fluorescent protein such as green fluorescent protein (GFP)which permits separation of cells expressing the protein using afluorescence activated cell sorter (FACS) machine, or using HOOKselection, available from Clontech). Alternatively, the marker canencode a polypeptide that is displayed on the cell surface anddetectable by a fluorescence-labeled antibody.

C. In Vitro Uses

The methods of the invention are useful for constructing cells and celllines that are useful for numerous purposes. Genetically modified cellscan be used to produce a desired gene product at a greater level thanotherwise produced by the cells, or a gene product that is modified fromthat otherwise produced. For example, one can modify a nonhuman cellgene that encodes a desired protein so that the amino acid sequence ofthe encoded protein corresponds to that of the human form of theprotein. Or the amino acid sequence can be changed to make the proteinmore active, more stable, have a longer therapeutic half-life, have adifferent glycosylation pattern, and the like. The methods can be usedto introduce a signal sequence at the amino terminus of a protein, whichcan facilitate purification of the protein by causing the cell tosecrete a protein that is normally not secreted.

As another example, one can use the methods of the invention to modifycells to make them express a polypeptide that, for example, is involvedin degradation of a toxic compound. If desired, expression can be madeinducible by the presence of the toxic compound. Such cells can be usedfor bioremediation of toxic waste streams and for cleanup ofcontaminated sites.

Cells that have been modified using the methods of the invention arealso useful for studying the effect of particular mutations. Forexample, one can disrupt expression of a particular gene and determinethe effect of that mutation on growth and/or development of the cell,and the interactions of the cell with other cells. Genes suspected ofinvolvement in disease, such as tumorigenesis (e.g., stimulators ofangiogenesis) and other diseases, can be disrupted to determine theeffect on disease development. Alternatively, expression ofdisease-related genes can be turned on or elevated and the effectevaluated.

Cells that are modified to express a particular gene under givenconditions can be used to screen for compounds that are capable ofinhibiting the expression of the gene. For instance, a cell can bemodified to place a gene required for cell growth under the control ofan inducible promoter. Test compounds are added to the growth mediumalong with the moiety that induces expression of the gene; cells in thepresence of a test compound that inhibits the interaction between theinducing moiety and the inducible promoter will not grow. Thus, thesecells provide a simple screening system for compounds that modulate geneexpression.

The invention also provides libraries of targeted integrants. Theselibraries are particularly suitable for use in the screening assaysdescribed above, as well as for genetic analyses. Many other uses forthe methods of the invention for introducing genetic mutations will beapparent to those of skill in the art.

D. Construction of Transgenic and Chimeric Animals

The invention also provides methods producing transgenic and chimericanimals, and transgenic and chimeric animals that are produced usingthese methods. A “chimeric animal” includes some cells that contain oneor more genomic modifications introduced using the methods and othercells that do not contain the modification. A “transgenic animal,” incontrast, is made up of cells that have all incorporated the specificmodification or modifications. While a transgenic animal is capable oftransmitting the modified target locus to its progeny, the ability of achimeric animal to transmit the modification depends upon whether themodified target locus is present in the animal's germ cells. Themodifications can include, for example, insertions, deletions, orsubstitutions of one or more nucleotides.

The methods of the invention are useful for producing transgenic andchimeric animals of most vertebrate species. Such species include, butare not limited to, nonhuman mammals, including rodents such as mice andrats, rabbits, ovines such as sheep and goats, porcines such as pigs,and bovines such as cattle and buffalo. Methods of obtaining transgenicanimals are described in, for example, Puhler, A., Ed., GeneticEngineering of Animals, VCH Publ., 1993; Murphy and Carter, Eds.,Transgenesis Techniques: Principles and Protocols (Methods in MolecularBiology, Vol. 18), 1993; and Pinkert, C A, Ed., Transgenic AnimalTechnology: A Laboratory Handbook, Academic Press, 1994. Transgenic fishhaving specific genetic modifications can also be made using the methodsof the invention. See, e.g., Iyengar et al. (1996) Transgenic Res. 5:147-166 for general methods of making transgenic fish.

One method of obtaining a transgenic or chimeric animal having specificmodifications in its genome is to contact fertilized oocytes with aparvoviral vector that includes a targeting construct that has thedesired modifications. For some animals, such as mice fertilization isperformed in vivo and fertilized ova are surgically removed. In otheranimals, particularly bovines, it is preferably to remove ova from liveor slaughterhouse animals and fertilize the ova in vitro. See DeBoer etal., WO 91/08216. In vitro fertilization permits the modifications to beintroduced into substantially synchronous cells. Fertilized oocytes arethen cultured in vitro until a pre-implantation embryo is obtainedcontaining about 16-150 cells. The 16-32 cell stage of an embryo isdescribed as a morula. Pre-implantation embryos containing more than 32cells are termed blastocysts. These embryos show the development of ablastocoel cavity, typically at the 64 cell stage. Embryos of greaterthan one cell can also be modified by introducing the recombinantparvoviral genomes of the invention. If desired, the presence of adesired modification in the embryo cells can be detected by methodsknown to those of skill in the art. Methods for culturing fertilizedoocytes to the pre-implantation stage are described by Gordon et al.(1984) Methods Enzymol. 101: 414; Hogan et al. Manipulation of the MouseEmbryo: A Laboratory Manual, C.S.H.L. N.Y. (1986) (mouse embryo); Hammeret al. (1985) Nature 315: 680 (rabbit and porcine embryos); Gandolfi etal. (1987) J. Reprod. Fert. 81: 23-28; Rexroad et al. (1988) J. Anim.Sci. 66: 947-953 (ovine embryos) and Eyestone et al. (1989) J. Reprod.Fert. 85: 715-720; Camous et al. (1984) J. Reprod. Fert. 72: 779-785;and Heyman et al. (1987) Theriogenology 27: 5968 (bovine embryos).Sometimes pre-implantation embryos are stored frozen for a periodpending implantation. Pre-implantation embryos are transferred to anappropriate female resulting in the birth of a transgenic or chimericanimal depending upon the stage of development when the transgene isintegrated. Chimeric mammals can be bred to form true germlinetransgenic animals.

Alternatively, the parvoviral vectors can be used to introduce specificgenetic modifications into embryonic stem cells (ES). These cells areobtained from preimplantation embryos cultured in vitro. See, e.g.,Hooper, M L, Embryonal Stem Cells: Introducing Planned Changes into theAnimal Germline (Modern Genetics, v. 1), Intl. Pub. Distrib., Inc.,1993; Bradley et al. (1984) Nature 309, 255-258. Transformed ES cellsare combined with blastocysts from a nonhuman animal. The ES cellscolonize the 20 embryo and in some embryos form the germ line of theresulting chimeric animal. See Jaenisch, Science, 240: 1468-1474 (1988).Alternatively, ES cells or somatic cells that can reconstitute anorganism (“somatic repopulating cells”) can be used as a source ofnuclei for transplantation into an enucleated fertilized oocyte givingrise to a transgenic mammal. See, e.g., Wilmut et al. (1997) Nature 385:810-813.

For production of transgenic animals containing two or more modifiedtarget loci, parvoviral vectors containing two targeting constructs canbe used, or more preferably two different parvoviral vectors, eachcontaining a different targeting construct, are introducedsimultaneously using the same procedure as for modifying a single targetlocus. Alternatively, each modification can be initially introduced intoseparate animals and then combined into the same genome by breeding theanimals. Or a first transgenic animal is produced that includes one ofthe desired modifications, after which the second modification isintroduced into fertilized ova or embryonic stem cells from that animal.

E. Ex Vivo Applications

The methods of the invention are useful for ex vivo applications, inwhich cells are removed from an organism, genetically modified using themethods, and reintroduced into an organism. In some applicationsgenetically modified cultured cell lines will be introduced into anorganism. The genetically modified cells can be introduced into the sameorganism from which the cells were originally obtained, or can beintroduced into a different organism of the same or a different species.Ex vivo therapy is useful, for example, in treating genetic diseasessuch as hemophilia and certain types of thalassemia, as well as otherdiseases that are characterized by a defect in a cell that can beremoved from the animal, modified using the methods of the invention,and reintroduced into the organism. The cells can be, for example,hematopoietic stem cells, which are derived from bone marrow or fetalcord blood, T-lymphocytes, B-lymphocytes, monocytes, liver cells, musclecells, fibroblasts, stromal cells, skin cells, or stem cells. The cellscan be cultured from a patient, or can be those stored in a cell bank(e.g., a blood bank). These methods are useful for treating humans, andalso for veterinary purposes.

The modified cells are administered to the animal or patient at a ratedetermined by the LD₅₀ of modified cell type, and the side-effects ofcell type at various concentrations, as applied to the mass and overallhealth of the patient. Administration can be accomplished via single ordivided doses.

Animal models and clinical protocols for ex vivo gene therapy have beenestablished for hematopoietic cells (Blaese et al. (1995) Science 270:475-480; Kohn et al. (1995) Nature Med. 1: 1017-1023), liver cells(Grossman et al. (1994) Nature Genet. 6: 335-341), muscle cells (Bonhamet al. (1996) Human Gene Ther. 7: 1423-1429), skin cells (Choate et al.(1996) Nature Med. 2: 1263-1267) and fibroblasts (Palmer et al. (1989)Blood 73: 438-445).

F. In Vivo Therapy

The methods of the invention are useful for correcting genetic defectsin vivo. Muscular dystrophy is just one example of a genetic diseasethat is often the result of one or a few mutations that result in anabnormal polypeptide being expressed that is unable to carry out itsfunction properly. The precise mutations for many variants of these andother genetic diseases are known to those of skill in the art, as aremethods for identifying undesirable genetic mutations. Examples include,but are not limited to, Charcot-Marie-tooth disease, Coffin-Lowrysyndrome, cystic fibrosis, fragile x syndrome, hemophilia, hereditarythrombotic predisposition (Factor V mutation) Huntington's disease,medium-chain acyl-coemzyme a dehydrogenase deficiency (mead), myotonicdystrophy, neurofibromatosis (nfl), sickle cell disease and globin chainvariations, spinal muscular atrophy, spincocerebellar ataxia, α and βthalassemia, von Hippel-Lindau disease, and the like. Genetic diseasesare reviewed in, for example, Shaw, D J (Ed.), Molecular Genetics ofHuman Inherited Disease, John Wiley & Sons, 1995; Davies and Read,Molecular Basis of Inherited Disease, 2^(nd) Edition, IRL Press, 1992.Human genetic diseases are treatable using the methods of the invention,as are those of other vertebrates. Non-genetic diseases can also betreated by manipulating genes. For example, one can modify a co-receptorfor HIV so that the receptor is no longer able to bind to HIV particles.

The parvoviral vectors containing recombinant parvoviral genomes can beadministered directly to the organism for modification of cells in vivo.Administration can be by any of the routes normally used for introducingviral vectors into ultimate contact with blood or tissue cells. Theviral vectors used in the present inventive method are administered inany suitable manner, preferably with pharmaceutically acceptablecarriers. Suitable methods of administering such viral vectors in thecontext of the present invention to a patient are available, and,although more than one route can be used to administer a particularviral vector, a particular route can often provide a more immediate andmore effective reaction than another route.

Pharmaceutically acceptable carriers are determined in part by theparticular viral vector being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of the pharmaceutical compositions ofthe present invention.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the vector dissolved indiluents, such as water, saline or PEG 400; (b) capsules, sachets ortablets, each containing a predetermined amount of the activeingredient, as liquids, solids, granules or gelatin; (c) suspensions inan appropriate liquid; and (d) suitable emulsions. Tablet forms caninclude one or more of lactose, sucrose, mannitol, sorbitol, calciumphosphates, corn starch, potato starch, tragacanth, microcrystallinecellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellosesodium, talc, magnesium stearate, stearic acid, and other excipients,colorants, fillers, binders, diluents, buffering agents, moisteningagents, preservatives, flavoring agents, dyes, disintegrating agents,and pharmaceutically compatible carriers. Lozenge forms can comprise theactive ingredient in a flavor, usually sucrose and acacia or tragacanth,as well as pastilles comprising the active ingredient in an inert base,such as gelatin and glycerin or sucrose and acacia emulsions, gels, andthe like containing, in addition to the viral vector, carriers known inthe art.

The viral vector, alone or in combination with other suitablecomponents, can be made into aerosol formulations to be administered viainhalation. Because the bronchial passageways are the usual route ofchoice for certain viruses, corresponding vectors are appropriatelyadministered by this method. Aerosol formulations can be placed intopressurized acceptable propellants, such as dichlorodifluoromethane,propane, nitrogen, and the like.

Suitable formulations for rectal administration include, for example,suppositories, which consist of the active viral vector with asuppository base. Suitable suppository bases include natural orsynthetic triglycerides or paraffin hydrocarbons. In addition, it isalso possible to use gelatin rectal capsules which consist of acombination of the viral vector with a base, including, for example,liquid triglyercides, polyethylene glycols, and paraffin hydrocarbons.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, intrathecal (in the cerebrospinal fluid),and subcutaneous routes, include aqueous and non-aqueous, isotonicsterile injection solutions, which can contain antioxidants, buffers,bacteriostats, and solutes that render the formulation isotonic with theblood of the intended recipient, and aqueous and non-aqueous sterilesuspensions that can include suspending agents, solubilizers, thickeningagents, stabilizers, and preservatives. The formulations can bepresented in unit-dose or multi-dose sealed containers, such as ampulesand vials, and in some embodiments, can be stored in a freeze-dried(lyophilized) condition requiring only the addition of the sterileliquid carrier, for example, water, for injections, immediately prior touse. Extemporaneous injection solutions and suspensions can be preparedfrom sterile powders, granules, and tablets of the kind previouslydescribed.

The dose administered to a patient, in the context of the presentinvention should be sufficient to effect a beneficial therapeuticresponse in the patient over time. The dose will be determined by theefficacy of the particular viral vector employed and the condition ofthe patient or animal, as well as the body weight or surface area of thepatient to be treated. The size of the dose also will be determined bythe existence, nature, and extent of any adverse side-effects thataccompany the administration of a particular vector or modified celltype in a particular patient or animal.

In determining the effective amount of the viral vector to beadministered in the treatment or prophylaxis of a particular disease,the physician or veterinarian needs to evaluate circulating plasmalevels, vector toxicities, and progression of the disease.

In the practice of this invention, the parvoviral vectors can beadministered, for example, by aerosolization and inhalation, intravenousinfusion, orally, topically, intramuscularly, intraperitoneally,intravesically or intrathecally. The preferred method of administrationwill often be intravenous or by inhalation, but the parvoviral vectorscan be applied in a suitable vehicle for the local and topical treatmentof virally-mediated conditions.

For administration, parvoviral vectors and genetically modified celltypes of the present invention can be administered at a rate determinedby the LD₅₀ of the parvoviral vectors, and the side-effects of theparvoviral vector or cell type at various concentrations, as applied tothe mass and overall health of the patient. Administration can beaccomplished via single or divided doses.

Protocols for in vivo gene therapy using adeno-associated viral vectorshave been described for the brain (Alexander et al. (1996) Human GeneTher. 7: 841-850), liver (Koeberl et al. (1997) Proc. Nat'l. Acad. Sci.USA 94: 1426-1431), lung (Flotte et al. (1993) Proc. Nat'l. Acad. Sci.USA 90: 10613-10617), and muscle (Xiao et al. (1996) J. Virol. 70:8098-8108). These methods can be adapted to other target organs by thoseof skill in the art.

G. Systems for Assaying Efficiency of Gene Targeting

The invention also provides methods for determining the efficiency ofparvoviral vector-mediated gene targeting. The methods involve using aretroviral vector to introduce into a target cell a mutant reporter genethat is subsequently corrected by gene targeting. The use of aretroviral vector to introduce the gene that is to be corrected hassignificant advantages over previously used assay systems. By placingthe mutant reporter gene within a retroviral vector that becomesintegrated into the target cell genome, the reporter gene becomesintegrated into the cellular genome within a well-characterizedchromosomal region (the integrated provirus). Because of this uniformityof the regions that surround the reporter gene, one can compare genetargeting efficiencies among a wide variety of cell types, includingprimary cells. Moreover, the retroviral vector proviruses can beintroduced into cells as single copy targets by infecting the cells at alow multiplicity of infection. An additional advantage is that, one canuse in the assays a polyclonal population of cells that contain targetloci at thousands of different retroviral vector integration sites, thustaking into account variations in gene targeting rates due to positioneffects.

The reporter genes that are introduced into the target cells using theretroviral vectors are defective in that the detectable and/orselectable gene product of the reporter gene is not expressed, absentcorrection by gene targeting. For example, the reporter gene can bedefective by virtue of a mutation in the coding region or in thepromoter or other sequence that controls expression of the reportergene. In presently preferred embodiments, the retroviral vectorsinclude, in addition to the defective reporter gene, a selectablemarker. Thus, one can select for cells that contain the integratedretroviral provirus. These cells are then used in the gene targetingassays.

Reporter genes that are suitable for use in the assays of the inventionare known to those of skill in the art. Typically, the reporter genesencode a polypeptide that is directly detectable, e.g., a fluorescentpolypeptide, or a polypeptide that is readily detectable through use ofdetection agents. For example, a reporter gene can encode an enzymethat, when present, converts a substrate into a readily detectable form.Alternatively, one can detect the reporter gene product through adetection agent that binds to the reporter gene product, for example, alabeled antibody. Suitable reporter genes include, but are not limitedto, genes that encode β-glucuronidase (GUS, uidA) from E. coli,β-galactosidase, luciferase (LUC) from firefly, and green fluorescentprotein (GFP) from jellyfish (see, e.g., Chalfie et al. (1994) Science263:802-805; Crameri et al. (1996) Nature Biotechnol. 14: 315-319;Chalfie et al. (1995) Photochem. Photobiol. 62: 651-656; Olson et al.(1995) J. Cell. Biol. 130: 639-650) and related antigens, several ofwhich are commercially available. In some embodiments, the reporter genewill encode a polypeptide that is expressed on the surface of the cells.One can then identify and enrich for those cells that have undergonesuccessful gene targeting by, for example, flow cytometry-based cellsorting.

The reporter gene can also be a selectable marker that allows selectionof modified cells that have undergone the desired gene targeting event.These genes can encode a gene product, such as a protein, necessary forthe survival or growth of transformed host cells grown in a selectiveculture medium. Host cells that do not express the gene product of theselection gene will not survive in the culture medium. Typical selectiongenes encode proteins that confer resistance to antibiotics or othertoxins, such as ampicillin, neomycin, kanamycin, chloramphenicol, ortetracycline. Examples of suitable coding sequences for selectablemarkers are: the neo gene which codes for the enzyme neomycinphosphotransferase which confers resistance to the antibioticskanamycin, neomycin, and G418 (Beck et al. (1982) Gene 19:327); the hyggene, which codes for the enzyme hygromycin phosphotransferase andconfers resistance to the antibiotic hygromycin (Gritz and Davies (1983)Gene 25:179). Alternatively, selectable markers can encode proteins thatcomplement auxotrophic deficiencies or supply critical nutrients notavailable from complex media. A number of selectable markers are knownto those of skill in the art and are described for instance in Sambrooket al., supra.

The assays are conducted by introducing into the cells that contain theintegrated retroviral provirus and associated defective reporter gene arecombinant parvoviral vector that includes the reporter gene that isdefective due to a different mutation than that in the retroviralprovirus. The defective reporter gene present in the parvoviral vectorcan be, for example, a full-length reporter gene that includes amutation that prevents expression of an active reporter gene product(e.g., a mutation in the promoter, coding region, or other region thataffects gene expression). Alternatively, the parvoviral vector caninclude only a subsequence of the reporter gene that is present in theretroviral provirus. In either case, the reporter gene present in theparvoviral vector overlaps the mutated region of the defective reportergene in the retroviral provirus. The parvoviral vector reporter gene ismutated at a different location than the provirus reporter gene, so thatthe reporter gene product is produced only by those cells in whichhomologous pairing and gene repair has occurred between the twodefective reporter genes.

The efficiency of gene targeting is then ascertained by determining thepercentage of cells that express the reporter gene product.

In additional embodiments, the invention provides methods to enrich forcells in which gene targeting has occurred. These methods are based onthe finding that a cell which undergoes gene targeting at one locus ismore likely to undergo targeting at a second locus. Accordingly, twotargeting constructs are introduced into cells. One targeting constructcan correct a defective reporter gene that, upon correction, produces areadily detectable reporter gene product. The other targeting constructis directed to the gene of interest. The two targeting constructs can bepresent on the same parvoviral vector, but more commonly each will becontained on a separate parvoviral vector. After introduction of thetargeting constructs into the cells, the cells are first screened orselected to identify those that express the reporter gene product. Theresulting enriched cell populations are then screened to identify thosecells in which the gene of interest has also undergone successful genetargeting.

The methods of enriching for target cells are useful for manyapplications. For example, germ cells, eggs, or other cells from which atransgenic organism can be reconstituted can be enriched for those cellsthat have undergone gene targeting. These cells are then screened toidentify those that contain a desired alteration at the target locus ofinterest. Cells that have undergone the desired gene targeting event arethen used to produce transgenic and/or chimeric animals.

EXAMPLES

The following examples are offered to illustrate, but not to limit thepresent invention.

Experimental procedures

A. Cell Culture

HeLa (Scherer et al. (1953) J. Exp. Med. 97: 695-709), HT-1080 (Rasheedet al. (1974) Cancer 33: 1027-33), and 293 (Graham et al. (1977) J. Gen.Virol. 36, 59-74) cells were cultured in Dulbecco's modified Eaglemedium (DMEM) with 10% heat-inactivated (56° C. for 30 minutes) fetalbovine serum (HyClone, Logan, Utah), 1.25 μg/ml amphotericin, 100 U/mlof penicillin, and 100 μg/ml of streptomycin at 37° C. in a 10% CO₂atmosphere. HT-1080 cells were maintained in HAT medium (DMEM containing13.61 μg/ml hypoxanthine, 0.176 μg/ml aminopterin and 3.875 μg/mlthymidine prior to their use in transduction experiments to minimize thenumber of HPRT cells in the population.

HSNO39 cells were created by cotransfection of HeLa cells with a BamH Ifragment containing the SV40 replication origin and promoter, mutant neogene and p15A origin (the same fragment present in pASNO39; see FIG. 1Aand below), and a BstYI fragment of pLHL containing the Moloney murineleukemia virus long terminal repeat promoter and hygromycin resistancegene. Transfected cells were selected for by growth in 0.2 mg/mlhygromycin (Calbiochem, San Diego, Calif.). HSNO39 cells were derivedfrom a single hygromycin-resistant colony and shown by Southern analysisto contain 3 copies of the neo gene per cell. HSNO39 cells were culturedin medium containing 0.2 mg/ml hygromycin prior to their use intransduction experiments.

B. Plasmids

The plasmids pAAV/ad (Samulski et al. (1989) J. Virol. 63:3822-8),pACYC184 (Chang et al., (1978) J. Bacteriol. 134: 1141-56), pBluescript(Stratagene, La Jolla, Calif.), psub201 (Samulski et al. (1987) J.Virol. 61: 3096-101), pSV2neo (Southern and Berg (1982) J. Mol. Appl.Genet. 1: 327-41) and pTR (Ryan et al., (1996) J. Virol. 70: 1542-53)have been described. pLHL was a gift from A. D. Miller (Fred HutchisonCancer Research Center, Seattle Wash.). pRepCap2 contains the XbaIfragment of psub201 containing the AAV2 rep and cap genes in the XbaIsite of pBluescript. pASNori2 was constructed by inserting a BamHI-Esp3Ineo fragment of pSV2neo containing a SspI-Bst11071 origin fragment frompACYC184 in the BstBI site (end-filled with Klenow fragment of DNAPolymerase I) downstream of the neo gene in to the Bg/II sites of theAAV vector backbone of pTR after attaching BamHI linkers to the pSV2neoEsp3I site. This same neo fragment was also used to construct the HSNO39cell line. pASNO39 is identical to pASNori2 except for a SalI linker(5-CGGTCGACCG; SEQ ID NO:6) in the end-filled Eagl site. pASNO648 isidentical to pASNori2 except for an end-filled and relegated Cspl site.pAHPe2/3 contains by 14, 057-17809 of the human HPRT locus (GenBankHUMHPRTB) in the BglII site of pTR as determined by DNA sequencing.pAHPe2/3X is identical to pAHPe2/3 except for an end-filled andrelegated XhoI site. Both orientations of HPRT sequences relative to thepTR backbone were obtained and no differences were noted in the genecorrection rates of the corresponding vectors. Human HPRT sequences werefrom the H&J lambda phage previously described (Patel et al. (1986) Mol.Cell. Biol. 6: 393-403).

C. Vector Production

AAV vector stocks were prepared as follows. 293 cells were plated at adensity of 4×10⁶ cells/dish in 24 dishes (10 cm). The next day each dishwas infected with 5.6×10⁷ plaque-forming units of adenovirus type 5(ATCC VR-5) and two hours later cotransfected with 4 μg of vectorplasmid and 16 μg of helper plasmid by the calcium phosphate method(Sambrook, supra.). After 3 days the cells and medium were harvested,freeze-thawed 3 times, clarified by centrifugation at 5800×g (5500 rpm)in a Sorvall HS4 rotor for 30 min. at 4° C., digested with 68 units/mlof micrococcal nuclease (Pharmacia, Piscataway, N.J.) at 37° C. for 1hour, treated with 50 ng/ml of trypsin at 37° C. for 30 min., andcentrifuged through 40% sucrose in phosphate buffered saline in aBeckman SW28 rotor at 27,000 rpm for 16 hours at 4° C. The pellets wereresuspended in 8 ml of a 0.51 g/ml solution of CsCl and centrifuged in aBeckman SW41 rotor at 35,000 rpm for 20 hours at 4° C. The region of thegradient containing AAV virions was collected, dialyzed against DMEMthrough a 50,000 molecular weight cutoff membrane (Spectrum, Houston,Tex.) and concentrated by centrifugation in Centricon 100 filters(Amicon, Inc., Beverly, Mass.). The vector plasmids used were pASNori2for AAV-SNori, pASNO648 for AAV-SNO648, pASNO39 for AAV-SNO39, pAHPe2/3for AAV-HPe2/3 and pAHPe2/3X for AAV-HPe2/3X. The helper plasmids usedwere pAAV/Ad (Samulski et al. (1989) supra.) or pRepCap2, which producedequivalent stock titers.

The titer of each vector stock was determined by Southern blots ofalkaline gels as follows. Ten μl stock dilutions were mixed with 2 μl of10% SDS, heated to 100° C. for 10 minutes, electrophoresed through 1.2%alkaline agarose gels (Sambrook, supra.), blotted onto Hybon-N membranes(Amersham, Buckinghamshire, England) and probed for vector sequences.The amount of full-length linear vector DNA present in each sample wasdetermined by comparison to standards present on the same gel using aMolecular Dynamics PhosphorImager 400S (Sunnyvale, Calif.), and thenumber of vector genomes per ml of stock calculated from thismeasurement. The same assay was used to locate vector particles on CsClgradients by electrophoresing 10 μl of each gradient fraction. Thenumber of intact vector genomes per ml of stock was the value used forvector particle numbers, which were typically >10¹¹/ml.

D. DNA Techniques

Enzymes were obtained from New England BioLabs, (Beverly, Mass.)Boehringer Mannheim, (Indianapolis, Ind.) or Stratagene (La Jolla,Calif.) and reactions were performed by using the manufacturersrecommended conditions. Plasmid construction, DNA purification, Southernblot analysis and bacterial culture were performed by standardprocedures (Sambrook et al., supra.). Plasmids were prepared by usingQiagen columns (Chatsworth, Calif.). Dye terminator cycle sequencing wascarried out using the ABI PRISM sequencing kit (Perkin Elmer, FosterCity, Calif.) and analyzed on an Applied Biosystems Inc. sequencer(Foster City, Calif.). Oligonucleotides were from Cruachem, Inc.(Dulles, Va.).

Integrated neo genes were rescued from transduced HSNO39 cells bydigesting high molecular weight genomic DNA calf intestinal phosphataseto prevent ligation of free ends in the sample, heat inactivated,extracted with phenol and chloroform, and precipitated with ethanol. Theresuspended DNA was digested with BamHI, extracted with phenol andchloroform and precipitated with ethanol. The resulting DNA fragmentswere resuspended, circularized with of T4 DNA ligase at 14° C. overnightand transferred to E. coli by electroporation or high efficiencychemical transformation. Bacterial colonies were selected for by growthon kanamycin plates.

Sequencing of the by 39 and by 648 mutations of corrected neo genesrecovered as bacterial plasmids was performed with primers 9606D(dATGGCTTTCTTGCCGCCA) (SEQ ID NO:1) and 9607A (dATACGCTTGATCCGGCTAC)(SEQ ID NO:2) respectively. HPRT exon 3 sequences were amplified fromhigh molecular weight genomic DNA by using a modification of apreviously published procedure (Rossiter et al. (1991) “Detection ofdeletions and point mutations.” In PCR. A practical approach, M. J.McPherson et al., eds. (Oxford, England: IRL Press), pp. 67-83) asfollows. PCR was performed on 100 ng of genomic DNA in 20 μl reactionvolume containing 2.1 picomoles of both 5 primer(dCCTTATGAAACATGAGGGCAAAGG) (SEQ ID NO:3) and 3 primer(TGTGACACAGGCAGACTGTGGATC) (SEQ ID NO:4), 6 mM MgSO₄, 1.25 mM eachdeoxynucleoside triphosphate, and 0.4 units Vent DNA Polymerase (NewEngland Biolabs, Beverly, Mass.). The reaction was carried out in aPTC-200 thermocycler (MJ Research, Watertown, Mass.) with denaturationat 94° C. for 4.5 minutes, followed by 30 cycles of 94° C. for 30seconds, 61° C. for 50 seconds and 72° C. for 2 minutes, then a finalpolymerization at 72° C. for 5 minutes. Six μl of the product wasfurther amplified in a 100 μl volume under the same conditions for 20cycles, and the PCR product was purified using a QIAquick kit (Qiagen,Chatsworth, Calif.) following the manufacturers protocol, and 75 ng ofthe purified product was used for DNA sequencing with the primerdACCTACTGTTGCCACTA (SEQ ID NO:5).

E. Gene Targeting Assays

Standard transduction experiments were performed by plating 5×10³ or1×10⁴ HSNO39 cells/well respectively into 96 (Nunc, Naperville, Ill.) or48 (Costar, Cambridge, Mass.) well plates or 2×10⁴ HT-1080 cells into 48well plates on day 1. On day 2, the medium was changed and vector stock(prepared in DMEM) was added to the well. The MOI was calculatedassuming one cell doubling since the original plating. On day 3, eachwell was treated with trypsin, and the cells were plated into 10 cmdishes. On day 4, the assays differed for each cell line.

For neo gene correction experiments, 90%, 9.5% and 0.5% of the cellsfrom each well were plated into different dishes. On day 4, G418 (1mg/ml active compound) was added to the 90% and 9.5% dishes andselection was continued for 10-12 days with medium changes every 3-4days. G418 was not added to the 0.5% dishes which served as a controlfor the total number of colony-forming units (CFU) from each originalwell. The colonies present in each dish were counted after staining withCoomassie brilliant blue G. The neo gene correction rate was calculatedas the number of G418-resistant CFU/total CFU for each original well.

For HPRT experiments, all the cells from each well were cultured withoutselection for 10-14 days after being plated into 10 cm dishes on day 3,to allow for elimination of existing HPRT protein in HPRT cells. Nosignificant differences were noted in HPRT mutation rates after 10 dayor 14 day culture periods. The medium was changed every 3-4 days andwhen dishes became too dense the cells were treated with trypsin anddilutions were plated into new dishes. After this phenotypic expressionperiod, 10⁵, 10⁴ and 10² cells of each culture were plated into new 10cm dishes, and the following day 6TG (5 μg/ml) was added to the 10⁵ and10⁴ cell dishes. 6TG selection was not applied to the 10² cell dishes asthese were used to calculate plating efficiencies. The cells werecultured for 10 additional days, stained with Coomassie brilliant blueG, and the surviving colonies were counted. The percent of 6TG-resistantCFU was determined after correcting for plating efficiencies.

Example 1 Correction of Mutant Neo Genes Using Adeno-Associated ViralVectors

This Example demonstrates that vectors based on adeno-associated virus(AAV) can efficiently modify specific chromosomal target sequences inhuman cells.

We used the selectable neomycin phosphotransferase gene (neo) as amarker to study gene correction by transduction. The vectors constructedfor these experiments were based on the AAV shuttle vector AAV-SNori(FIG. 1A), which contains the neo gene under the control of both thebacterial Tn5 promoter and SV40 early promoters, and the p15A plasmidreplication origin, which supports stable replication in Escherichiacoli (Cozzarelli et al. (1968) Proc. Nat'l. Acad. Sci. USA 60: 992-999).The AAV2 terminal repeats flank these internal sequences and contain allthe cis-acting sequences required for replication and packaging of thevector genome (McLaughlin et al. 15 (1988) J. Virol. 62: 1963-1973;Samulski et al., supra.). Mammalian cells transduced by AAV-SNori areresistant to G418, and the integrated proviruses can be recovered asbacterial plasmids expressing kanamycin resistance. Mutations wereintroduced into the AAV-SNori vector at by 39 (a 14 nucleotideinsertion) and by 648 (a 3 nucleotide insertion) of the neo gene (bp 1being the translation start codon), to generate the vectors AAV-SNO39and AAV-SNO648. Both mutations disrupt neo gene function, but genecorrection between the two mutant genes can regenerate a functional geneand confer G418 resistance.

HeLa cells were used as a model human system to study gene correction byAAV vectors. A HeLa cell line containing integrated copies of theinternal portion of the AAV-SNO39 genome (lacking the terminal repeats)was created by cotransfection of this fragment with a hygromycinselectable marker (see Experimental Procedures). Severalhygromycin-resistant clones were isolated and screened for the presencethe mutant neo gene cassette by Southern analysis. One cell line,designated HSNO39, appeared to contain 3 intact copies per cell of theneo cassette integrated at different locations and was chosen forfurther experiments.

A. Frequency of Neo Gene Correction

HSNO39 cells were infected with AAV-SNO648 vector stocks, treated withtrypsin and plated at different dilutions on the following day, thenselected in G418 two days after infection. Dilutions were also grownwithout selection to determine the total number of colony-forming unitsin the sample. Correction of the mutant chromosomal neo genes byincoming vector genomes was measured as the fraction of coloniesresistant to G418. As shown in Table 1, approximately 0.1% of HSNO39cells were resistant to G418 after infection with AAV-SNO648. Thisrepresents a minimal neo gene correction rate as some cells couldcontain silenced genes with inadequate expression levels. Infection ofHeLa cells with AAV-SNO648 did not produce G418-resistant colonies,demonstrating that reversion of the by 648 mutation in the vector didnot occur at detectable rates. Similarly, G418-resistant colonies werenot detected in uninfected HSNO39 cells or HSNO39 cells infected withAAV-SNO39, showing that reversion of the chromosomal by 39 mutation didnot occur. About 0.6% of HeLa cells were resistant to G418 aftertransduction with the AAV-SNori vector, which contains a functional neogene and can integrate at random chromosomal locations by non-homologousrecombination. Thus the neo gene correction rate was about 5-fold lowerthan the random vector integration rate of a similar vector.

TABLE 1 Neo Gene Correction Cell Line Vector/Plasmic: 1 MOI FractionG418R HSNO39 none — <5.3 × 10−5 ″ ″ —  <4.3 × 1 OT5 ″ ″ — <4.3 × 105  ″″ — <1.4 × 10−5 HSNO39 AAV-SNO648 40,000 9.6 × 104 ″ ″ 40,000  6.7 ×10−4 ″ ″ 400,000  2.0 × 10−3 ″ ″ 400,000  1.4 × 10−3 HeLa AAV-SNO64840,000 <6.0 × 10−5 ″ ″ 40,000 <5.7 × 10−5 ″ ″ 400,000 <6.8 × 10−5 ″ ″400,000 <6.6 × 10−5 HSNO39 AAV-SNO39 375,000 <6.3 × 10−5 1,500,000 <6.6× 10−5 HeLa AAV-SNori 100,000  7.3 × 10−3 ″ ″ 100,000  5.3 × 10−3

B. Structure of the Chromosomal Neo Genes

Several G418-resistant colonies obtained by infecting HSNO39 cells withAAV-SNO648 were isolated, expanded to approximately 2×10⁷ cells, andanalyzed by Southern blots. After digestion with BamHI, genomic DNA fromthe parental HSNO39 cells contained 3 major neo-hybridizing bands of2.7, 5.0 and >20 kb, representing the three integrated copies of the neogene cassette (FIG. 1B). A 2.7 kb BamHI neo fragment was used togenerate the HSNO39 line by cotransfection. A faint 8.0 kb band was alsoobserved at less than one copy per cell, and may be due to methylationor mutation at one of the BamHI sites in a subset of HSNO39 cells. Fourout of eleven HSNO39/AAV-SNO648 G418-resistant clones (1, 2, 9 and 10)contained the same 3 major bands as the parental line, with noadditional fragments. The 8.0 kb band of clones 4 and 5 could representthe faint band of the same size in the parental line. New bands wereobserved in 4 of the clones, suggesting that random vector integrationhad also occurred in a subset of cells exposed to the vector. Threeclones were missing bands present in the parental line (3, 4 and 7),which can be explained by modification of a BamHI site rather thanrearranged neo cassettes, as no novel bands were observed in theseclones. Homology between the vector and chromosomal neo cassettesextends up to the BamHI site, so modification of the chromosomalsequence at this site by vector DNA could have destroyed the site. Theseresults demonstrate that the majority of G418-resistant clones isolatedcontained at least one corrected neo gene without additionalrearrangements due to vector integration.

C. Sequence of the Corrected Neo Genes

To assess the fidelity of the gene correction process we recoveredseveral corrected neo genes in bacterial plasmids and sequenced therelevant regions. The neo gene cassette present in HSNO39 cells and theAAV-SNO648 vector can replicate and confer kanamycin resistance in E.coli (FIG. 1A), allowing us to recover corrected neo genes as bacterialplasmids. Chromosomal DNA from the G418-resistant HSNO39/AAV-SNO648clones shown in FIG. 1B was digested with BamHI, circularized with DNAligase, and transferred to bacteria that were then selected forkanamycin resistance. As shown in Table 2, corrected neo genes wererecovered as bacterial plasmids from 7/11 clones. It is possible thatmore persistent attempts to recover plasmids from the remaining 4 cloneswould also have been successful. Plasmids isolated from these bacteriawere digested with BamHI and only those with a unique site wereconsidered correct. A 2.7 kb plasmid was recovered from each of theseven clones that by restriction digestion appeared to be a circularizedBamHI fragment identical to that used to produce the HSNO39 line, exceptfor the absence of the by 39 mutation. A second 20 kb plasmid was alsorecovered from clone 11, which appeared to correspond to the highmolecular weight band observed on Southern blots. Apparently at leasttwo of the neo genes present in this cell line had been corrected. Therecovered plasmids contained wild type neo genes based on digestion withBsiEI, which can identify the by 39 and by 648 mutations (see FIG. 1A).

TABLE 2 Rescue of Corrected Neo Genes KanR Col- Cell onies Re- FractionPlasmid Line Southern Results covered Correct Sizes HSNO39 2.7, 5.0,(8.0), >20 kb 0 — — Clone 1 no change 14 11/12 2.7 kb Clone 2 no change7 6/6 2.7 kb Clone 3 02.7 kb 0 — — Clone 4 05.0 kb 0 — — Clone 5 +5.5 kb7 6/7 2.7 kb Clone 6 +(2.4, 5.2) kb 3 2/3 2.7 kb Clone 7 02.7 kb 0 — —Clone 8 +18 kb 6 5/6 2.7 kb Clone 9 no change 2 2/2 2.7 kb Clone 10 nochange 0 — — Clone 11 +6.6 kb 4 4/4 2.7, 20 kb  

We sequenced the regions surrounding the by 39 and by 648 mutations ofeach recovered plasmid. More than 200 by of sequence was obtained fromeach region and in all cases the sequence corresponded exactly to thatof the wild-type neo gene. Thus the gene correction process led to anaccurate deletion of the 14 nucleotide insertion present at thechromosomal by 39 mutation, without additional genetic changes andwithout insertion of the by 648 mutation present in the vector. However,because our assay required the presence of a functional neo gene, anyadditional mutations created during the targeting event that disruptedneo gene function would have been excluded from our analysis.

Example 2

Modification of the Human HPRT Gene by AAV Vectors

We also studied gene correction by AAV vectors at the human hypoxanthinephosphoribosyltransferase locus (HPRT). The HPRT gene is frequently usedto study mutation because HPRT cells can be selected for by growth inthe presence of 6-thioguanine (6TG), so mutagenesis at the single copyX-linked locus can be measured in diploid male cells. We used HT-1080human fibrosarcoma cells to study gene targeting at the HPRT genebecause this cell line has a pseudodiploid male karyotype (Rasheed etal. (1974) Cancer 33: 1027-1033) and has been used previously in HPRTgene targeting experiments (Pikaart et al. (1992) Mol. Cell. Biol. 12:5785-92; Zheng et al. (1991) Proc. Nat'l. Acad. Sci. USA 88: 8067-71).

AAV vectors containing a region of the human HPRT locus encompassingexons 2 and 3 were used to introduce a specific mutation into the HPRTgene of HT-1080 cells (FIG. 2A). The AAV-HPe2/3 vector contains wildtype genomic sequence, while the AAV-HPe2/3X vector contains a 4nucleotide insertion in exon 3, which causes a frameshift in the HPRTcoding sequence. HT-1080 cells were infected with both vectors andselected for 6TG resistance after culturing the cells for a periodwithout selection to allow for elimination of existing HPRT protein (seeExperimental Procedures). As shown in FIG. 3, about 1/2000 HT-1080 cellsinfected with the mutant AAV-HPe2/3X vector were 6TG-resistant. Thisrepresents the minimum HPRT gene modification frequency, as HT-1080cells are not uniformly diploid and could contain additional Xchromosomes (Rasheed et al., supra.). The AAV-HPe2/3X vector targetingfrequency was about 30 fold above the background mutation rate.Infection with the wild-type vector did not raise the HPRT mutation rateabove background levels.

Southern analysis of several 6TG-resistant clones isolated afterinfection with AAV-HPe2/3X confirmed that the vector mutation had beenintroduced into the chromosomal HPRT locus. FIG. 2B shows the results ofdigestion with HindIII, which cuts outside of vector sequences andproduces a 6.8 kb chromosomal fragment containing exons 2 and 3 inHT-1080 cells. This band was unaltered in all of the clones analyzed,demonstrating the absence of major rearrangements in this region. Fivelines contained additional bands, presumably due to random vectorintegration. Further digestion with PvuI showed that 10/13 clonescontained the 2.2 kb band expected from transfer of the vector PvuI siteinsertion mutation to the chromosome (FIG. 2C). To date we have analyzed24 independent 6TG-resistant HT-1080 clones infected with AAV-HPe2/3X,18 of which had the expected Pvul site insertion in exon 3 as determinedby Southern analysis. We used the polymerase chain reaction (PCR) toamplify exon 3 from the genomic DNA of 6 of these clones and sequencedthe PCR products (see Experimental Procedures). At least 330 by ofunambiguous sequence was obtained from each clone, including all of exon3. In all cases the entire sequence was identical to the published HPRTsequence except for the predicted 4 nucleotide insertion in exon 3.Clones without additional vector integration events were sequenced toavoid amplification of unlinked vector DNA. Sequence from the parentalHT-1080 cell line did not contain this insertion mutation.

Example 3 The Effects of Vector Dose on Gene Correction

The mutant neo gene in HSNO39 cells was corrected with the AAV-SNO648vector using a range of infection multiplicities. FIG. 4 shows theresults of several experiments plotted as infecting vector genomes percell versus the percent of cells with corrected neo genes. The genecorrection rate increased from about 0.001 to greater than 0.1 percentwith increasing vector doses of 40 to 2×10⁶ vector particles per cell.These results suggest that the gene correction reaction is limited bythe number of vector molecules entering the cell.

Example 4 Comparison of Transduction and Transfection Gene CorrectionRates

We compared neo gene correction rates in HSNO39 cells transduced with 25AAV-SNO648 vector stocks or transfected with the plasmid pASNO648, whichcontains the entire AAV-SNO648 genome (FIG. 5). The transduction ratewas at least 400 times that obtained by transfection. Furthertransfection experiments using the pASNO39 plasmid, which contains thesame mutation as the HSNO39 cell line, gave similar results to pASNO648,suggesting that the gene correction rate of pASNO648 was actually due toreversion rather than homologous pairing. Thus the homologous pairingrate by transduction could have been much more than 400 times thatobtained by transfection. One potential explanation for thesedifferences is that plasmid uptake occurs in only a small proportion oftransfected cells, while vector genomes presumably enter every cell. Thestable transfection efficiency of HSNO39 cells was approximately 7% asdetermined by transfections with pASNori2, which is identical topASNO648 except it contains a functional neo gene. Presumably, an evenhigher percentage of cells were transiently transfected. Even aftermaking the conservative assumptions that 7% of transfected cellscontained functional plasmid molecules, and that all the G418-resistantcolonies obtained by transfecting pASNO648 were due to homologouspairing, the gene correction rate is still 30 fold higher in transducedcells than that observed in the subpopulation of cells that incorporatedplasmid DNA (1.7×10⁻³ vs 5.7×10⁻⁵).

Example 5 Modification of HPRT Genes in Normal Human Fibroblasts

Standard transduction experiments were performed by plating 5×10⁴ normalhuman fibroblasts per well into 24 well plates. On day two, the mediumwas changed and vector stock (AAV-HPe2/3 or AAV-Hpe2/3X, prepared inDMEM) was added to the well. On day three, each well was treated withtrypsin and the cells were plated into 10 cm dishes. On day four, all ofthe cells from each well were cultured without selection for 12-14 daysto allow for elimination of existing HPRT protein in HPRT cells. Themedium was changed every 3-4 days and when cells became too dense thecells were treated with trypsin and dilutions were plated into newdishes. After this phenotypic expression period, 10⁵, 10⁴, and 10² cellsof each culture were plated into new 10 cm dishes, and the following day6TG (10 Tg/ml) was added to the 10⁵ and 10⁴ cell dishes. 6TG selectionwas not applied to the 102 cell dishes, as these were used to calculateplating efficiencies. The cells were cultured for ten additional days,stained with Coomassie brilliant blue G, and the surviving colonies werecounted. The percentage of 6TG-resistant colony-forming units wasdetermined after correcting for plating efficiencies. Four differentnormal fibroblast lines were studied: MHF1, MHF2 and MHF3 were fromnormal males, and FHF1 was from a normal female.

As shown in FIG. 6, modification of the HPRT gene was proportional tothe number of infecting viral genomes per cell. Modifications could beintroduced into the HPRT genes of all four fibroblast lines (FIG. 7).

Summary of Examples 1-5

These Examples demonstrates that vectors based on adeno-associated virus(AAV) efficiently and specifically modify vertebrate chromosomal targetsequences. Both integrated neomycin phosphotransferase genes and thenormal, X-linked hypoxanthine phosphoribosyltransferase gene weretargeted by AAV vectors. Site-specific genetic modifications could beintroduced into >0.1% of the total cell population, a significantlyhigher rate than could be achieved by transfection, and themodifications could be introduced into normal primary cells. Themajority of modified cells contained no other detectable geneticchanges, and DNA sequencing demonstrated the high fidelity of theprocess. These results suggest that parvoviral vectors are useful forintroducing specific genetic changes into the genomic DNA of a widevariety of vertebrate cells.

Example 6 Assay System for Determining Efficiency of Gene Targeting

This Example describes a system for determining the efficiency of genetargeting. The system makes use of a bicistronic retroviral vector tointroduce a target locus into the cells. The strategy employed isillustrated in FIG. 8.

A. Correction of Alkaline Phosphatase Genes

A human placental alkaline phosphatase (AP) reporter gene was used inthis experiment. The LAPSN retroviral vector contains the AP gene underthe control of the murine leukemia virus (MLV) LTR, and a neomycinresistance gene (neo) under the control of an internal SV40 earlypromoter (Miller et al. (1994) Proc. Nat'l. Acad. Sci. USA 91: 78-82).This vector was engineered to contain either a 4 by deletion atnucleotide 375 of the AP reading frame to create the vectorLAP375(Δ4)SN, or a 2 by deletion at nucleotide 961 to create LA961(Δ2)SN(FIG. 8). Retroviral vector stocks were prepared by transienttransfection of PG13 cells, with pseudotype vector particles in thegibbon ape leukemia virus (GALV) envelope (Miller et al. (1991) J.Virol. 65: 2220-2224). This pseudotype allows for efficient infection ofhuman cells. Normal, primary human fibroblasts were transduced withLAP375(Δ4)SN/PG13 and selected in G418. A polyclonal population derivedfrom more than 5,000 independent transduced cells was obtained, each ofwhich presumably contained a different, single copy integration site ofthe retroviral target locus.

An AAV vector, AAV-5′APBss, was prepared that contained the 5′ portionof the AP gene (a 2486 by BssH II restriction fragment from pLAPSN; seeFIG. 9). The fibroblast population containing LAP375(Δ4)SN was infectedwith the AAV-5′APBss vector at an MOI of 1,200 vector particles percell, and the cells were cultured and stained for AP expression atdifferent times after infection.

As seen in FIG. 10, the number of AP⁺ cell foci increased during theculture period, and the larger AP⁺ foci only appeared at later times asexpected. The number of small foci consisting of 1-2 AP⁺ (presumablyreflecting recent gene targeting events) also increased with time,suggesting that gene correction continued to occur over the entireculture period. This is not surprising, as AAV vector genomes persistfor several days as episomal molecules in human fibroblasts (Russell etal. (1994) Proc. Nat'l. Acad. Sci. USA 91: 8915-8919). AP⁺ cells werenot observed among cells that did not contain the retroviral target, noramong those cells that did not receive the AAV vector. These controlsrule out reversion mutation as a source of AP positivity. The absolutenumbers of gene targeting events were similar to those obtained in HPRTtargeting experiments using normal human fibroblasts at equivalent MOIs(Russell and Hirata (1998) Nat. Genet. 18: 325-330), which wererelatively low in this experiment. Thus, these results demonstrate thatthe retroviral target system can be used in normal human cells as aconvenient assay for measuring gene correction rates by AAV vectors.

B. Correction and Rescue of Neo Genes

In this experiment, gene correction at mutant neo genes in normal humancells was examined. The retroviral vector MLV-LHSNO39 was used tointroduce mutant neo genes into normal human fibroblasts. MLV-LHSNO39contains a functional hygromycin resistance gene and a neo gene with aninsertion mutation at by 39 of the coding sequence. The vector alsocontains a p15A bacterial plasmid replication origin and a prokaryoticpromoter controlling the neo gene, which allowed us to recover correctedproviruses as bacterial plasmids that confer resistance to kanamycin orneomycin.

Normal human fibroblasts were transduced with MLV-LHSNO39, andhygromycin-resistant cells were selected. These cells were thentransduced with the AAV vector AAV-SNO648, which has about 2.7 kb ofsequence identity with the MLV

LHSNO39 vector, except that the neo gene is mutated at by 648 of thecoding sequence and there is no mutation at by 39 (FIG. 11). Both neomutations disrupt neo-function, so G418-resistant cells must haveundergone a gene targeting event.

After infection with the AAV-SNO648 vector, approximately 1 in 1000normal human fibroblasts containing the LHSNO39 target provirus becameG418 resistant. This gene correction rate is similar to what wasobserved in HeLa cells containing the same neo mutation in Example 1above.

Several G418-resistant colonies were isolated and expanded toapproximately 10⁶-10⁷ cells for DNA analysis (Table 3). Genomic DNA fromeach clone was digested with EcoRI, which digests the LHSNO39 targetsonce in the hygromycin resistance gene outside the region of homologywith the AAV-SNO648 vector (FIG. 11). Genomic DNA fragments containingthe corrected neo extend downstream to the next EcoRI site, which liesin the flanking chromosomal DNA, so they include the entire regionhomologous to the AAV-SNO648 vector, as well as regions just outside theregion of homology to AAV-SNO648, and the junction between theretroviral provirus and chromosomal DNA. These fragments werecircularized with DNA ligase and recovered in bacteria as plasmidsconferring kanamycin or neomycin resistance. Table 3 summarizes therestriction digestion and sequence analysis to date of these recoveredplasmids.

TABLE 3 Analysis of Corrected neo Genes Recovered as Bacterial PlasmidsKan- Resistant Plasmids Restriction Sequence Re- Size (kb) AnalysisAnalysis G418- covered/ of Left Right Left Right Resistant μg FlankingHomol- Homol- Homol- Homol- Fibroblast Genomic Genomic ogy ogy ogy ogyClone DNA DNA End End End End 1 210 1.0 Rear- Intact +14 by ND ranged 2<2 NA NA NA NA NA 3 203 0.8 Intact Intact Intact ND 4 497 0.5 IntactIntact Intact ND 5 117 0.5 Intact Intact Intact ND 6 167 0.3 IntactIntact Intact ND 7 23 9.0 Intact Intact Intact ND 8 110 3.0 IntactIntact Intact ND 9 <2 NA NA NA NA NA 10 200 6.5 Intact Intact Intact ND11 7 0.8 Intact Intact Intact ND 12 120 1.2 Intact Intact Intact ND ND =not determined; NA = not applicable

Of twelve independent G418-resistant fibroblast clones, ten containedcorrected neo targets that could easily be recovered as bacterialplasmids. There was some variability in the efficiency of plasmidrescue, which presumably was related to differences in flankingchromosomal DNA sequences. In each case, a different plasmid wasrecovered, confirming that the retroviral proviruses were integrated atdifferent chromosomal locations as expected. These rescued plasmids weredigested with a panel of enzymes designed to determine if any majorrearrangements occurred during the targeting process, especially at theleft and right homology ends where the sequence of the AAV targetingvector diverges from that of the retroviral target. Based on restrictionanalysis, one of ten recovered targets had a rearrangement at the lefthomology end, and all ten of the right homology ends appeared intact.Sequencing confirmed that nine of ten rescued plasmids had nomodifications at the left homology ends. Sequencing of the plasmid fromclone #1 demonstrated that a rearrangement had occurred, which was inpart due to an insertion of 14 additional non-homologous nucleotidesfrom the AAV vector.

These rescue experiments and the sequence analysis of HPRT genetargeting (Example 2) demonstrate that AAV-mediated gene correction isusually a high fidelity process consisting of an accurate replacement ofsequences at the intended modification site. In addition, the processdoes not usually introduce secondary mutations or rearrangements, evenat the ends of homology. In the case of conventional gene targetingexperiments based on electroporation, some studies (but not others(Thomas et al. (1992) Mol. Cell. Biol. 12: 2919-2923; Zheng et al.(1991) Proc. Nat'l. Acad. Sci. USA 88: 8067-8071) have suggested thatsecondary mutations can occur at the targeted locus (Brinster et al.(1989) Proc. Nat'l. Acad. Sci. USA 86: 7087-7091; Thomas and Capecchi(1986) Nature 324: 34-38), especially rearrangements at the homologyends (Doetschman et al. (1988) Proc. Nat'l. Acad. Sci. USA 85:8583-8587; Hasty et al. (1991) Mol. Cell. Biol. 11: 4509-4517), so itwas important to determine the fidelity of the AAV-mediated reaction. Itshould also be noted that we have never observed duplications atAAV-targeted loci, which are often produced by conventional genetargeting methods using insertion vectors.

These neo rescue experiments demonstrate several important points.Because the fibroblast population being studied contains manyindependent target loci at random chromosomal positions, we can concludethat AAV-mediated gene correction is not limited to particular loci(excluding any potential site preferences for retroviral integration).The fact that the process is a high fidelity gene correction event iscrucial for use of this method for therapeutic purposes. In addition,this demonstration with neo targets allows one to more confidentlyinterpret data with other retroviral targets such as the AP gene(above), where sequence analysis is more difficult. These studies alsodemonstrate the experimental potential of the retroviral targetcorrection system, especially that targeted loci can be routinelyrecovered and analyzed as bacterial plasmids from normal human cells.Therefore, these experiments are readily adaptable to a wide variety ofcell types and culture conditions which may require sequence analysis oftargeted loci, not only to demonstrate fidelity but also to determinewhich mutations were introduced during gene correction.

C. Gene Correction in Other Cell Lines

A key feature of the retroviral target gene correction system describedherein is its ability to be used in different cell types. This allowsone to compare gene targeting rates at the same locus with the samevector in cell lines with various mutations in genes that may beimportant for gene targeting. As both the retroviral vectors used tointroduce the targets and the AAV targeting vectors have broad hostranges, a large number of mutant cell lines can be studied. Aparticularly relevant set of cells are the primary human fibroblastswith mutations in genes involved in DNA repair and/or recombination, asthese same genes could play a role in gene targeting. Many of these celltypes are available from the Coriell Institute for Medical Research(Camden N.J.).

In this Example, four different primary human fibroblast cultures wereused. These fibroblasts were isolated from normal males (MHF2), orpatients with xeroderma pigmentosum (XP) complementation groups A (XPA1)or C(XPC1) and Bloom's Syndrome (BSI). Both diseases are associated withdefects in DNA repair, with XP patients exhibiting increased sensitivityto ultraviolet light and Bloom's Syndrome patients havinghypermutability.

In order to measure gene correction rates we used the alkalinephosphatase (AP) gene targeting system shown in FIG. 9. All fourfibroblast types were first transduced with the retroviral vectorsLAP375(44)SN or LAP961(42)SN containing mutant AP genes and polyclonalG418-resistant populations were selected that consisted of more than5,000 independent proviral integration events. These transducedfibroblasts were then infected with the AAV-5′APBss targeting vector,cultured for 8 days, and stained for AP expression. The gene correctionrate was calculated as the number of AP⁺ foci per 10⁵ infected cells. NoAP⁺ foci were observed in cultures that did not receive vector, nor incultures that did not contain the retroviral vector target sites,confirming that AP expression was due to AAV-mediated gene correction.As shown in FIG. 12, there was little difference in gene targeting ratesbetween the XP fibroblasts XPA1 and XPC1 as 30 compared to the normalhuman fibroblasts MHF2. However, the Bloom's Syndrome fibroblasts BS1has an approximately 5-fold higher gene correction rate with bothretroviral targets. Since cells from Bloom's Syndrome patients are knownto have increased rates of sister chromatid exchange (Chaganti et al.(1974) Proc. Nat'l. Acad. Sci. USA 71: 4508-4512), this suggests thatgene targeting and sister chromatid exchange may involve similarmechanisms.

These experiments demonstrate that the retroviral target correctionassay can be used on different cell lines, including primary fibroblastcultures. We have also used the system on HT-1080 human fibrosarcomacells and HeLa cells, indicating that the methods are applicable to awide range of cell types.

Example 7 In Vivo Correction of Chromosomal Genes

This Example describes methods for performing gene correction in vivousing the gene targeting methods of the invention. Two murine models areused, one with a mutation in the endogenous 11-glucuronidase gene(11-gus) and the other with engineered mutations in13-galactosidase(f-gal) transgenes.

A. β-Gus Gene Correction In Vivo

A murine model of mucopolysaccharidosis type VII (MPS VII) is caused bya single base pair deletion in the β-gus gene (Sands and Birkenmeier(1993) Proc. Nat'l. Acad. Sci. USA 90: 6567-6571). Mice homozygous forthe MPS VII mutation gusinPs develop a lysosomal storage disease withcharacteristics similar to human MPS VII or Sly syndrome, including ashortened life span, skeletal abnormalities, and an accumulation ofglycosaminoglycans in various tissues. These mice are an ideal model tostudy in vivo gene targeting for the following reasons. First, both thewild type and mutant genomic loci have been cloned and sequenced(Gallagher et al. (1987) Genomics 1: 145-152; Sands and Burkenmeier,supra.). Second, by using a histochemical stain (similar to staining forβ-gal), gus⁺ cells can easily be distinguished from gus^(mps) cells inboth tissue culture and tissue sections from several organs. Third, abiochemical assay for β-glucuronidase is available (Gallagher (1992). APCR assay has been developed that distinguishes between mutant and wildtype alleles (Wolfe and Sands (1996) Murine mucopolysaccharidosis typeVII: a model system for somatic gene therapy of the central nervoussystem. In Protocols for gene transfer in neuroscience: towards genetherapy of neurological disorders, P. R. Lowenstein and L. W. Enquist,eds.; John Wiley and Sons, Ltd.). Transformed fibroblast cell lines fromgus^(mps) heterozygotes and homozygotes can be used for in vitrostudies. Moreover, the mutation is a small (1 bp) deletion that isreadily amenable to correction by AAV vectors.

The structure of the mouse β-gus genomic locus and AAV vectors that aresuitable for use in the experiments are shown in FIG. 13. The AAVvectors contain approximately 4 kb of genomic DNA, including exon 10.The AAV-Gus10w5 vector is made from wild-type DNA, while the controlvector AAV-Gus10mps contains the 1 by deletion in exon 10 that isresponsible for MPS VII.

In initial experiments, the gus^(mps) mutation is corrected intransformed fibroblasts from MPS VII mice in vitro, by infecting withthe AAV-Gus10wt vector and then staining for β-gus expression. Cellsinfected with the AAV-Gus10mps vector will serve as a control. If genecorrection occurs, β-gus expression is observed only in cells infectedwith the wild-type vector. As cultured mouse cells are generally moredifficult to transduce with AAV vectors than mouse cells in vivo, thisexperiment is expected to provide a reasonable expectation that thevectors will also function in the in vivo experiments.

In vivo experiments are then performed on homozygous gus^(mps) miceobtained by breeding heterozygotes and identified by PCR for gus^(mps)alleles (Wolfe and Sands, supra.). The liver is used as a target tissuebecause β-gus is expressed at high levels in this organ. Moreover, AAVvectors are readily delivered to the liver (Koeberl et al. (1997) Proc.Nat'l. Acad. Sci. USA 94: 1426-1431; Snyder et al. (1997) Nat. Genet.16: 270-276). Eight to 10 week old mice receive 10¹⁰ to 10¹¹ vectorparticles, either by intravenous or intrahepatic injection. Previousgene addition studies indicate that both types of injection willefficiently deliver vector particles to the liver (Koeberl et al.,supra.).

Mice are analyzed for β-gus gene correction at different times afterinfection (approximately 2 weeks, 2 months, and/or at the time ofdeath). Based on the time course of AP gene targeting with AAV vectors(FIG. 10) and previous gene addition experiments using AAV vectors todeliver genes to the mouse liver (Snyder et al., supra.), it may takeseveral days or weeks to reach a maximum gene correction level. Thelater time points will assess the persistence of gene expression fromcorrected alleles.

The analysis of injected mice can consist of staining tissue sectionsfor β-gus expression, measuring β-glucuronidase activity in tissuehomogenates, and isolating DNA from tissue samples for Southern analysisand PCR. The tissues to be examined can include liver, spleen, kidney,lung and brain. Individual gus⁺ cells should be clearly visible instained sections, allowing one to derive a gene correction rate based onestimates of the total cell number in the section or visualized field. Aclose examination of stained sections may also allow one to identifywhich types of cells are expressing β-gus. This histochemical analysisis expected to be the most sensitive assay for gene correction, with theability to detect gene correction rates far below 1%. Based on genetargeting rates in vitro, the in vivo gene targeting rates are expectedto be 0.1-1.0%. A comparison of sections from mice injected withAAV-Gus10wt and the control vector AAV-Gus10mps will demonstrate thatthe observed β-gus expression is due to specific correction of thegus^(mps) by deletion mutation. Measurements of β-glucuronidase levelsin tissue homogenates will also be performed to help determine the genecorrection rate.

If the gene correction rate approaches 10%, one can most likely identifycorrected chromosomal genes directly in Southern blots, since thewild-type allele can be digested by N1aIV, but the mutant allele cannot.Where the gene correction rate is lower, Southern analysis of genomicDNA samples can be performed in order to identify randomly integratedvector proviruses that may be present in addition to targeted loci(using restriction enzymes and/or probes from the AAV terminal repeats).

PCR analysis can also be used to identify corrected alleles. Forexample, one can amplify exon 10 using primers at the positions shown inFIG. 13. These PCR products should only be produced from genomic β-gusloci and not from randomly integrated vectors. Digesting the productswith N1aIV will then distinguish corrected from mutant alleles, andshould be able to detect even a relatively small fraction of wild-type,corrected alleles.

Similar gene correction experiments are also performed on newbornanimals by injecting vectors into the superficial temporal vein (Sandset al. (1993) Lab. Invest. 68: 676-686). This approach has proven verysuccessful in gene addition experiments with AAV vectors and shouldallow for vector delivery at a time when more cell proliferation isoccurring. In addition, any therapeutic effect that may be due to genecorrection must take place before the permanent damage that takes placein older animals. One can follow the development of the animals comparedto uncorrected gus^(mps) littermates and establish by microscopytechniques that a reduction in distended lysosomes has occurred (Wolfeand Sands, supra.).

B. β-Gal Transgene Correction In Vivo

A transgenic mouse model is also developed for in vivo gene correctionexperiments. This model is based on a β-gal shuttle transgene that canbe rescued from mammalian cells as a bacterial plasmid that allowslac-bacteria to grow in media containing lactose. Transgenic micecontaining mutated versions of the β-gal shuttle transgene are infectedwith AAV targeting vectors that can correct the β-gal mutation, andcells with corrected transgenes are detected by histochemical stainingof tissue sections, similar to the experiments described above for β-gusgene correction. The major advantage of the β-gal system is that thecorrected genes can be rescued in bacteria, providing an independentestimate of correction rates and allowing sequence analysis of targetedgenes. In addition, by selecting mice with desirable transgeneexpression profiles, it is possible to perform gene correctionexperiments in a variety of different organs. The approach is similar toother transgenic animal models that screen for mutagenesis at β-galtransgenes (Gossen et al. (1994) Mutat. Res. 307: 451-459), except thatin this case one is assaying for gene correction.

FIG. 14 shows constructs that can be used for these experiments (inlinear form). pCnZSNO contains a nuclear-localizing β-gal gene under thecontrol of the strong cytomegalovirus (CMV) promoter, a neo gene underthe control of the SV40 early promoter, and a p15A plasmid origin. TheSV-neo-p15A portion of the plasmid is the same shuttle vector cassetteused in Example 6, which can replicate and confer kanamycin resistancein E. coli. The β-gene also contains a bacterial lac operator andpromoter (“lac” in FIG. 14), which controls transcription in E. coli,allowing blue/white screening for corrected alleles and providinganother prokaryotic selectable marker that confers lactose-dependentgrowth to lac bacteria. HeLa cells transfected with pCnZSNO formG418-resistant colonies with blue nuclei when stained for β-galexpression. Bacterial colonies can be selected for pCnZSNO by growing inmedia containing kanamycin or lactose as a sole sugar source.

Plasmid pCnZSNO can be further modified to contain additional markergenes that aid in identifying transfected cells expressing β-galtransgenes. Since mutations are to be introduced into β-gal for genecorrection experiments, an additional marker for identification isnecessary. For example, one can insert downstream reporter genes thatare controlled by the same promoter as β-gal, but initiate translationfrom an internal ribosome entry site (IRES) derived fromencephalomyocarditis virus. Plasmids with green fluorescent protein(GFP) and/or alkaline phosphatase (AP) can be constructed, for example,as shown in FIG. 14.

The pCnZGSNO and pCnZAPSNO plasmids are first tested in cultured cells.Mutations that disrupt protein function are engineered into the plasmidβ-gal genes. HeLa cells are transfected with these mutated plasmids andstable integrants are selected in G418. Individual G418-resistantcolonies are selected and tested for either GFP or AP expression byvisualization under fluorescent light or histochemical staining.Colonies 20 expressing GFP or AP are then screened for genecorrectability using an AAV vector that contains a wild type portion ofβ-gal (FIG. 14). The portion of β-gal included in the vector will notencode a functional β-gal protein. Corrected genes are identified bothby staining HeLa cells for β-gal expression (which is analogous to theAP gene correction assay described previously), and by rescuing plasmidsand screening bacterial colonies for β-gal expression.

The plasmid rescue experiments are similar to those described above, andconsist of digesting integrated transgenes outside of the requiredgenetic elements with restriction enzymes, circularizing the restrictionfragments, electroporating bacteria, and then selecting either forkanamycin resistance (neo rescue) or growth in lactose-containing media(β-gal rescue). In the case of neo rescue experiments, one can also growthe colonies on Xp-gal plates and measure the percentage of recoveredplasmids with corrected transgenes by blue/white colony screening. Thesein vitro experiments demonstrate that all the constructs required forgene correction are working properly.

Transgenic animals are then created by pronuclear injection of theappropriate pCnZGSNO or pCnZAPSNO constructs. Pups are screened for thepresence of —the transgene by assaying for GFP expression underfluorescent light or AP expression by histochemical staining of tail ortoe sections. Pups expressing the transgene are also analyzed bySouthern blots to determine the structure of the transgene. Thesefounders are bred and their offspring screened to determine which organsand tissues express the GFP or AP reporter genes. Presumably, the mutantβ-gal transcripts will also be highly expressed in these same tissues,since it is controlled by the same promoter. The goal is to identifyanimals that express high levels of GFP or AP in a wide variety oftissues. Such animals will be versatile models to study in vivo genecorrection.

Once the appropriate transgenic mice are identified, they are used forin vivo gene correction experiments. The AAV vectors to be used willcontain either wild type β-gal sequences or a version of β-gal with thesame mutation as present in the transgenic animal. Only the wild-typevector should result in gene correction. Eight to 10 week old mice areinoculated with 10¹⁰ to 10¹¹ AAV vector particles by intravenous orintrahepatic injection, similarly to the experiments described above.These injections are expected to deliver vector mainly to the liver(Koeberl et al., supra.). Intramuscular injections are also performed inthese transgenic animals, because AAV vectors have been found toefficiently transduce skeletal muscle in gene addition experiments(Fisher et al. (1997) Nat. Med. 3: 306-312; Xiao et al. (1996) J. Virol.70: 8098-8108), and the procedure is simple to perform. In addition,intravenous injections can be performed on newborn animals as describedabove.

Animals are sacrificed at different times after injection (approximately2 weeks, 2 months, and 12 months or at the time of death from othercauses) and analyzed by several methods. First, tissue sections arestained histochemically for nuclear β-gal expression to determine thegene correction rate in the relevant organs. Second, DNA analysis isperformed, including Southern blots and plasmid rescue in bacteria.Southern analysis is useful in determining the overall structure of thetarget locus and identifying random integration events that might alsohave occurred. Plasmid rescue is performed by selecting for either ofthe neo or β-gal genes, and the recovered plasmids are sequenced todemonstrate that gene correction occurred as predicted and to assess thefidelity of the reaction. The gene correction rate is also measured byblue/white staining of colonies for β-gal expression in plasmidsrecovered by neo gene selection.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference for allpurposes.

1. (canceled)
 2. A recombinant parvoviral vector for introducing agenetic modification at a preselected genomic target locus of avertebrate cell, the recombinant parvoviral vector comprising: atargeting construct which comprises a genomic DNA sequence which issubstantially identical to the genomic target locus except for themodification to be introduced, wherein the modification to be introducedis flanked by regions substantially identical to the genomic targetlocus, said regions being at least 100 nucleotides in length; and atleast one parvoviral inverted terminal repeat (ITR) flanking thetargeting construct.
 3. The recombinant parvoviral vector of claim 2wherein the targeting construct further comprises a DNA that isexogenous to the vertebrate cell, wherein the exogenous DNA ispreselected for modification of the genomic target locus.
 4. Therecombinant parvoviral vector of claim 3, wherein the exogenous DNAcomprises a selection marker that is functional in the vertebrate cell.5. The recombinant parvoviral vector of claim 3 wherein the modificationcomprises one or more deletions, insertions, substitutions, or acombination thereof.
 6. The recombinant parvoviral vector of claim 2,wherein the target locus comprises a DNA sequence selected from thegroup consisting of a transcriptional regulatory region, a splicesignal, a sequence involved in DNA replication, a matrix attachmentpoint, a chromosomal recombination hotspot, a structural gene, or acoding region for a signal sequence, and portions thereof.
 7. Therecombinant parvoviral vector of claim 6, wherein the DNA sequencecomprises a structural gene and the modification results in an aminoacid substitution, deletion, insertion, or a combination thereof, in apolypeptide encoded by the gene.
 8. The recombinant parvoviral vector ofclaim 6, wherein the DNA sequence comprises a transcriptional regulatoryregion selected from the group consisting of a promoter, an enhancer, aresponse element, a transcription termination signal, and a locuscontrol region.
 9. The recombinant parvoviral vector of claim 8, whereina gene under the control of the modified transcriptional regulatoryregion is expressed at a different level than that at which the gene isexpressed under equivalent conditions when the gene is under the controlof the unmodified transcriptional regulatory region.
 10. The recombinantparvoviral vector of claim 2 wherein the targeting construct includes arecombination signal that is flanked by the polynucleotide sequencesthat are substantially identical to the target locus.
 11. Therecombinant parvoviral vector of claim 10 wherein the recombinationsignal is one or more Lox sites.
 12. The recombinant parvoviral vectorof claim 2 wherein the targeting construct further comprises aselectable marker.
 13. The recombinant parvoviral vector of claim 12wherein the selectable marker comprises the neo gene or hyg gene. 14.The recombinant parvovirus vector of claim 2 wherein the targetingconstruct further comprises a screenable marker.
 15. The recombinantparvoviral vector of claim 14 wherein the screenable marker comprisesβ-galactosidase, β-glucuronidase, luciferase, alkaline phosphatase or afluorescent marker.
 16. The recombinant parvoviral vector of claim 2wherein the targeting construct further comprises a targeting enhancer.17. The recombinant parvoviral vector of claim 2 comprising twoparvoviral terminal repeats (ITR), one at each end of the targetingconstruct.
 18. The recombinant parvovirus vector of claim 2 wherein theparvoviral rep and cap genes have been deleted.
 19. The recombinantparvoviral vector of claim 2 wherein the parvovirus is AAV.
 20. Therecombinant parvoviral vector of claim 2 packaged in a parvorviralcapsid.
 21. The recombinant parvoviral vector of claim 2 comprising twotargeting constructs.
 22. The recombinant parvoviral vector of claim 21wherein the parvoviral capsid comprises one or more modifications toimprove cell targeting.
 23. The recombinant parvoviral vector of claim21, wherein the parvoviral capsid is an AAV capsid.
 24. The recombinantparvoviral vector of claim 2, wherein the parvoviral vectors foradministration to the cell consist essentially of either all plus or allminus strands.
 25. The recombinant parvoviral vector of claim 2, whereinthe vector is comprised within a parvoviral particle.